Detection and prognosis of cervical cancer

ABSTRACT

The present invention relates to methods and kits for identifying, diagnosing, prognosing, and monitoring cervical cancer. These methods include determining the methylation status or the expression levels of particular genes, or a combination thereof.

FIELD OF THE INVENTION

The present invention relates to the area of cancer diagnostics andtherapeutics. In particular, it relates to methods and kits foridentifying, diagnosing, prognosing, and monitoring cervical cancer.These methods include determining the methylation status or theexpression levels of particular genes, or a combination thereof.

BACKGROUND TO THE INVENTION

Cervical cancer is the fifth most deadly cancer in women. Worldwide,approximately 500,000 cases of cervical cancer are diagnosed and about250,000 women die from this disease annually(www.who.int/mediacentre/factsheets).

Most (80-90%) invasive cervical cancer develops in flat, scaly surfacecells that line the cervix (called squamous cell carcinomas, SCC).Approximately 10-15% of cases develop in glandular surface cells (calledadenocarcinomas, AdC). Less commonly, cervical cancers have features ofboth SCC and AdC. These are called adenosquamous carcinomas or mixedcarcinomas (www.cancer.org).

During the process of cervical cancer development, normal cervical cellsgradually develop pre-cancerous changes that turn into cancer. Cervicalcancer evolves from pre-existing noninvasive premalignant lesionsreferred to as cervical intraepithelial neoplasias (CINs), ranging fromCIN I (mild dysplasia) to CIN II (moderate dysplasia) to CIN III (severedysplasia/carcinoma in situ). This process usually takes several yearsbut sometimes can happen in less than a year. For most women,pre-cancerous cells will remain unchanged and disappear without anytreatment.

Screening for malignant and premalignant disorders of the cervix isusually performed according to the Papanicolaou (PAP) system. Thecervical smears are examined by light microscopy and the specimenscontaining morphologically abnormal cells are classified into PAP I toV, at a scale of increasing severity of the lesion. But, present PAPtest has some limitations and is not completely ideal for screening asit suffers from suboptimal single-test sensitivity, limitedreproducibility, and many equivocal.

There is a strong association between certain subtypes of the HumanPapillomavirus (HPV) and cervical cancer. Studies have shown that onlyhigh-risk HPV types are involved in the progression from cytologicalnormal cervix cells to high grade squamous intraepithelial lesions.Around 15 high-risk (cancer-causing) HPV types have been identified.Although it has been suggested that high-risk HPV testing may improvecervical cancer screening, the specificity for high grade cervicalneoplasia of high risk HPV testing is relatively low. This lowspecificity of HPV testing leads to a higher number of unnecessarilyfollow-up diagnostic workups (e.g. colposcopy) and unnecessarilytreatment with cryotherapy or loop electrosurgical excision procedure,which permanently alters the cervix and have unknown consequences onfertility and pregnancy.

To improve early detection, the combination of HPV and PAP tests is nowapproved by the FDA for screening women 30 years of age and older.However, co-testing substantially increases the cost of screening.

In the meanwhile, vaccines for preventing cervical cancer have beendeveloped and one has already been approved by the FDA. But,immunization will only protect against HPV types that are targeted bythe vaccine; protection will not be absolute and its longevity isuncertain; as yet, the possibility of genotype replacement cannot beexcluded; and older women not covered by vaccination programs willcontinue to be at risk. Therefore, cervical screening will still berequired for control.

Cancer biomarkers have been described in literature and aberrantmethylation of genes has been linked to cervical cancer (Virmani et al,2001). In addition, methylation markers may serve for predictivepurposes as they often reflect the sensitivity to therapy or duration ofpatient survival.

DNA methylation is a chemical modification of DNA performed by enzymescalled methyltransferases, in which a methyl group (m) is added tocertain cytosines (C) of DNA. This non-mutational (epigenetic) process(mC) is a critical factor in gene expression regulation. (See J. G.Herman, Seminars in Cancer Biology, 9: 359-67, 1999).

An early diagnosis is critical for the successful treatment of manytypes of cancer, including cervical cancer. If the exact methylationprofiles of cervical tumors are available and drugs targeting thespecific genes are obtainable, then the treatment of cervical cancercould be more focused and rational. Therefore, the detection and mappingof novel methylation markers is an essential step towards improvement ofcervical cancer prevention, screening, and treatment. Thus, there is acontinuing need in the art to identify methylation markers that can beused for improved assessment of cervical cancer.

SUMMARY OF THE INVENTION

The present invention is based on the finding that several genes areidentified as being differentially methylated in cervical cancers. Thisinformation is useful for cervical cancer screening, risk-assessment,prognosis, disease identification, disease staging, and identificationof therapeutic targets. The identification of new genes that aremethylated in cervical cancer allows accurate and effective earlydiagnostic assays, methylation profiling using multiple genes andidentification of new targets for therapeutic intervention.

Accordingly, in a first aspect, the invention provides a method foridentifying cervical cancer or its precursor, or predisposition tocervical cancer. Epigenetic modification of at least one gene selectedfrom the group consisting of genes according to Table 1, is detected ina test sample containing cervical cells or nucleic acids from cervicalcells. The test sample is identified as containing cells that areneoplastic, precursor to neoplastic, or predisposed to neoplasia, or ascontaining nucleic acids from cells that are neoplastic, precursor toneoplastic, or predisposed to neoplasia.

Preferably, the at least one gene is selected from a group of genesconsisting of JAM3, LMX1A, CDO1, NID2, ALX3, ALX4, AR, ARID4A, ATM,AURKA, B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf48, C9orf19, CALCA,CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU,COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4,EPB41L3, FOS, FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, GPNMB, GREM1,Gst-Pi, HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1,JPH3, KNDC1, KRAS, LAMA1, LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B,NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC,PRKCE, RAD23B, RALY, RARA, RASSF1A, RBP4, RECK, RPRM, SALL4, SEMA3F,SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST,TAC1, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1,WT1, XRCC3, and ZGPAT.

In one embodiment of the present invention, the detection of epigeneticmodification comprises detection of methylation of a CpG dinucleotidemotif in the gene and/or promoter region of the gene; and/or detectionof expression of mRNA of the gene.

The invention also relates to a kit for assessing cervical cancer or itsprecursor, or predisposition to cervical cancer in a test samplecontaining cervical cells or nucleic acids from cervical cells. The kitcomprises in a package: a reagent that (a) modifies methylated cytosineresidues but not non-methylated cytosine residues, or that (b) modifiesnon-methylated cytosine residues but not methylated cytosine residues;and at least one pair of oligonucleotide primers that specificallyhybridizes under amplification conditions to a region of a gene selectedfrom the group consisting of genes according to Table 1 and/or theaforementioned group of genes. The region is preferably within about 10kbp of said gene's transcription start site.

In a further aspect, the invention provides for oligonucleotide primersand/or probes and their sequences for use in the methods and assays ofthe invention.

The invention also relates to screening protocols for the screening ofwoman for cervical cancer and the precursors thereof. Such method forcervical cancer screening combines hr-HPV testing and methylationtesting, or combines PAP tests with methylation testing. Methylationtesting in such screening method preferably detects the epigeneticmodification of at least one gene selected from the group consisting ofJAM3, LMX1A, CDO1, NID2, ALX3, ALX4, AR, ARID4A, ATM, AURKA, B4GALT1,BMP2, BMP6, BNIP3, C13orf18, C16orf48, C9orf19, CALCA, CAMK4, CCNA1,CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU, COL1A1, CPT1C,CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4, EPB41L3, FOS,FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, GPNMB, GREM1, Gst-Pi, HHIP, HIN1,HOOK2, HOXA1, HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1, JPH3, KNDC1, KRAS,LAMA1, LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B, NDRG2, NOL4, NPTX1,NPTX2, OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY,RARA, RASSF1A, RBP4, RECK, RPRM, SALL4, SEMA3F, SLC5A8, SLIT1, SLIT2,SLIT3, SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST, TAC1, TERT, TFPI-2,TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1, WT1, XRCC3, and ZGPAT.Dependent on the outcome, the women screened for cervical cancer isreferred for colposcopy, or referred for hr-HPV and/or PAP testingand/or methylation testing on a more regular basis.

Epigenetic loss of gene function can be rescued by the use of DNAdemethylating agents and/or DNA methyltransferase inhibitors.Accordingly, the invention also provides for a method for predicting thelikelihood of successful treatment or resistance to treatment of cancerwith such agent. If the gene is methylated, the likelihood of successfultreatment is higher than if the gene is unmethylated, or methylated to alesser degree. Conversely, if the gene is unmethylated, or methylated toa lesser degree, the likelihood of resistance to treatment is higherthan if the gene is methylated.

In a related aspect, epigenetic loss of gene function(s) can identifythe stage of the disease and from that the need of treatment.Accordingly, the invention provides for a method for predicting suitabletreatment comprising determining the methylation status of a gene or acombination of genes. If the gene is methylated, the need of cervicalresection is identified; if the gene is unmethylated or methylated to alesser degree, it is decided that there is no need for cervicalresection.

SUMMARY OF THE FIGURES

FIGS. 1A, B, and C: The number of probes (w) that is retrieved usingparameters x (number of P-calls in primary cancers for probe), y (numberof P-calls in untreated cell-lines for probe) and z (number of P-callsin treated cell-lines for probe).

FIG. 2: Step-plot to determine optimal number of probes for furtheranalysis. Step-plot of the number of retrieved known markers as afunction of the position after relaxation ranking (this is the number ofselected probes after ranking). The step plot shows the actual(observed) number of markers. If the markers were randomly distributed,one would expect the profile, marked with ‘expected’ (details in thetext). The trend of the observed markers versus the number of selectedprobes is indicated with dashed lines.

FIG. 3: (Hyper) methylation analysis of the promoter region (−430 to −5of TSS) of the CCNA1 gene by COBRA and sequence analysis.

A: schematic representation of the restriction enzyme sites (B: BstUIand T: TaqI) in the virtual hypermethylated BSP nucleotide sequenceafter bisulfate treatment. Vertical bars represent CG site, arrowrepresents TSS (retrieved from Ensembl).

B: Result of COBRA analysis of the BSP products of 10 tumor samples(T1-T10), in vitro methylated DNA as a positive control (IV) andleukocyte DNA as a negative (unmethylated) control (L).

C: Schematic representation of the sequencing results. From each tumor,the BSP-products were cloned into TOPO-pCR4 (Invitrogen) and sequencing(BaseClear) was performed on M13-PCR products of 7-9 independent clones.Circles represent CG dinucleotides: the darker, the more clones at thissite were methylated.

FIG. 4: Representative COBRA on 3 gene promoters (SST, AUTS2 and SYCP3).

A: schematic representation of the restriction enzyme sites in thevirtual hypermethylated BSP nucleotide sequence after bisulfatetreatment. (B: BstUI, T: TaqI and H: HinfI). Bars represent CG site andarrow is TSS (retrieved from Ensembl).

B: Result of COBRA analysis of BSP products of tumor samples (T1-T10)and 5 normal cervices (N1-N5), in vitro methylated DNA as a positivecontrol (IV) and leukocyte DNA as a negative (unmethylated) control (L);lane B is water blank.

FIG. 5:

A: Position of the different primers relative to the TSS (transcriptionstart site). Multiple primer designs are displayed by blue boxes and redboxes (=final primer pairs retained for the assays). The exon of ALX4 isindicated in green. The number of CpG count is spotted in blue over aregion of 20 Kb.

B: List of sequences for the different primer sets, converted andunconverted amplicon sequences used in FIG. 5 A.

FIGS. 6A and B: Ranked methylation table from the Lightcycler platform.27 methylation profiles from cervical cancer samples (left) are comparedagainst 20 normal tissue samples (right). Samples are shown along theX-axis where each vertical column represents the methylation profile ofone individual sample across the 63 different assays (Y-axis). Assaysdemonstrating the best methylation discriminators between the 2 groupsare displayed at the top, with discrimination effect decreasing towardsthe bottom. The black boxes indicate the methylated results; grey boxesindicate the unmethylated results; white boxes indicate invalid results.(NA: not applicable; NT: not tested)

FIG. 7: Amplification plot for the standard curve for TAC1_56187

FIG. 8: Amplification plot for standard curve and samples for TAC1_56187

FIG. 9: Linear regression of standard curve for TAC1_56187

FIG. 10: Decision tree for ratio determination

FIG. 11: Performance of the individual markers on cervical tissuesamples using qMSP.

DETAILED DESCRIPTION OF THE INVENTION

We describe a new sorting methodology to enrich for genes which aresilenced by promoter methylation in human cervical cancer. Thepharmacological unmasking expression microarray approach is an elegantmethod to enrich for genes that are silenced and re-expressed duringfunctional reversal of DNA methylation upon treatment with demethylatingagents. However, such experiments are performed in in vitro (cancer)cell lines mostly with poor relevance when extrapolating to primarycancers. To overcome this problem, we incorporated data from primarycancer samples in the experimental design. A pharmacological unmaskingmicroarray approach was combined with microarray expression data ofprimary cancer samples. For the integration of data from both cell linesand primary cancers, we developed a novel ranking strategy, whichcombines reactivation in cell lines and no expression in primary cancertissue.

We also used a Genome-wide Promoter Alignment approach with the capacityto define a further substantial fraction of the cancer gene promoter CpGisland DNA methylome. Markers clustering with known methylation markersmight indicate towards common mechanisms underlying the methylationevent and thus identify novel genes that are more methylation-prone.

Studies of the genes defined by the different approaches will contributeto understanding the molecular pathways driving tumorigenesis, provideuseful new DNA methylation biomarkers to monitor cancer risk assessment,early diagnosis, and prognosis, and permit better monitoring of genere-expression during cancer prevention and/or therapy strategies.

Using the aforementioned techniques, we have identified cytosines withinCpG dinucleotides of DNA from particular genes isolated from a testsample, which are differentially methylated in human cervical cancertissue samples and normal cervical tissue control samples. The cancertissues samples are hypermethylated or hypomethylated with respect tothe normal samples (collectively termed epigenetic modification). Thedifferential methylation has been found in genomic DNA of at least onegene selected from the group consisting of JAM3, LMX1A, CDO1, NID2,ALX3, ALX4, AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, BMP6, BNIP3,C13orf18, C16orf48, C9orf19, CALCA, CAMK4, CCNA1, CCND2, CDH1, CDH4,CDK6, CDKN1B, CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, CYCLIND2,DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4, EPB41L3, FOS, FOXE1, GADD45A,GATA4, GDAP1L1, GNB4, GPNMB, GREM1, Gst-Pi, HHIP, HIN1, HOOK2, HOXA1,HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1, JPH3, KNDC1, KRAS, LAMA1,LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B, NDRG2, NOL4, NPTX1, NPTX2,OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, RARA,RASSF1A, RBP4, RECK, RPRM, SALL4, SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3,SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST, TAC1, TERT, TFPI-2, TLL1,TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1, WT1, XRCC3, and ZGPAT.

Accordingly, in a first aspect, the invention provides a method foridentifying cervical cancer or its precursor, or predisposition tocervical cancer. Epigenetic modification of at least one gene selectedfrom the group consisting of genes according to Table 1, is detected ina test sample containing cervical cells or nucleic acids from cervicalcells. The test sample is identified as containing cells that areneoplastic, precursor to neoplastic, or predisposed to neoplasia, or ascontaining nucleic acids from cells that are neoplastic, precursor toneoplastic, or predisposed to neoplasia.

Preferably, the at least one gene is selected from a group of genesconsisting of JAM3, LMX1A, CDO1, NID2, ALX3, ALX4, AR, ARID4A, ATM,AURKA, B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf48, C9orf19, CALCA,CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU,COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4,EPB41L3, FOS, FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, GPNMB, GREM1,Gst-Pi, HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1,JPH3, KNDC1, KRAS, LAMA1, LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B,NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC,PRKCE, RAD23B, RALY, RARA, RASSF1A, RBP4, RECK, RPRM, SALL4, SEMA3F,SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST,TAC1, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1,WT1, XRCC3, and ZGPAT.

Preferably, at least one gene is selected from the group consisting ofJAM3, LMX1A, CDO1, NID2, CCNA1, HOXA11, GREM1 and TAC1. Preferably,epigenetic silencing of a gene combination is detected and preferablyselected from the group of gene combinations consisting of:

-   -   NID2 and HOXA11;    -   JAM3, CDO1, HOXA11, and CCNA1;    -   JAM3 and HOXA11;    -   JAM3, HOXA11 and GREM1;    -   JAM3, NID2, HOXA11 and CDO1;    -   JAM3, TAC1, HOXA11, and CDO1;    -   JAM3, HOXA11, and CDO1;    -   JAM3 and CDO1;    -   JAM3 and NID2;    -   NID2 and CDO1;    -   JAM3 and LMX1A    -   NID2 and LMX1A, and    -   JAM3, CDO1 and NID2

“Identifying” a disease or predisposition of disease is defined hereinto include detecting by way of routine examination, screening for adisease or pre-stadia of a disease, monitoring staging and the stateand/or progression of the disease, checking for recurrence of diseasefollowing treatment and monitoring the success of a particulartreatment. The identification can also have prognostic value, and theprognostic value of the tests can be used as a marker of potentialsusceptibility to cancer.

The term “Epigenetic modification” can be described as a stablealteration in gene expression potential that takes place duringdevelopment and cell proliferation, mediated by mechanisms other thanalterations in the primary nucleotide sequence of a gene. Three relatedmechanisms that cause alteration in gene expression are recognized: DNAmethylation, histone code changes and RNA interference.

Epigenetic modification of a gene can be determined by any method knownin the art. One method is to determine that a gene which is expressed innormal cells or other control cells is less expressed or not expressedin tumor cells. Diminished gene expression can be assessed in terms ofDNA methylation status or in terms of expression levels as determined bytheir methylation status, generally manifested as hypermethylation.Conversely, a gene can be more highly expressed in tumor cells than incontrol cells in the case of hypomethylation. This method does not, onits own, however, indicate that the silencing or activation isepigenetic, as the mechanism of the silencing or activation could begenetic, for example, by somatic mutation. One method to determine thatsilencing is epigenetic is to treat with a reagent, such as DAC(5′-deazacytidine), or with a reagent which changes the histoneacetylation status of cellular DNA or any other treatment affectingepigenetic mechanisms present in cells, and observe that the silencingis reversed, i.e., that the expression of the gene is reactivated orrestored.

Another means to determine epigenetic modification is to determine thepresence of methylated CpG dinucleotide motifs in the silenced gene orthe absence of methylation CpG dinucleotide motifs in the activatedgene. In one embodiment, epigenetic modification of a CpG dinucleotidemotif in the promoter region of the at least one gene selected from agroup of genes according to Table 1 is determined. Methylation of a CpGisland at a promoter usually prevents expression of the gene. Theislands can surround the 5′ region of the coding region of the gene aswell as the 3′ region of the coding region. Thus, CpG islands can befound in multiple regions of a nucleic acid sequence. The term “region”when used in reference to a gene includes sequences upstream of codingsequences in a regulatory region including a promoter region, in thecoding regions (e.g., exons), downstream of coding regions in, forexample, enhancer regions, and in introns. All of these regions can beassessed to determine their methylation status. When the CpGdistribution in the promoter region is rather scarce, levels ofmethylation are assessed in the intron and/or exon regions. The regionof assessment can be a region that comprises both intron and exonsequences and thus overlaps both regions. Typically these reside nearthe transcription start site (TSS), for example, within about 10 kbp,within about 5 kbp, within about 3 kbp, within about 1 kbp, within about750 bp, within about 500 bp, within 200 bp or within 100 bp. Once a genehas been identified as the target of epigenetic modification in tumorcells, determination of reduced or enhanced expression can be used as anindicator of epigenetic modification.

Expression of a gene can be assessed using any means known in the art.Typically expression is assessed and compared in test samples andcontrol samples which may be normal, non-malignant cells. Either mRNA orprotein can be measured. Methods employing hybridization to nucleic acidprobes can be employed for measuring specific mRNAs. Such methodsinclude using nucleic acid probe arrays (e.g. microarray technology, insitu hybridization, Northern blots). Messenger RNA can also be assessedusing amplification techniques, such as RT-PCR. Sequencing-based methodsare an alternative; these methods started with the use of expressedsequence tags (ESTs), and now include methods based on short tags, suchas serial analysis of gene expression (SAGE) and massively parallelsignature sequencing (MPSS). Differential display techniques provideanother means of analyzing gene expression; this family of techniques isbased on random amplification of cDNA fragments generated by restrictiondigestion, and bands that differ between two tissues identify cDNAs ofinterest. Specific proteins can be assessed using any convenient methodincluding immunoassays and immuno-cytochemistry but are not limited tothat. Most such methods will employ antibodies, or engineeredequivalents thereof, which are specific for the particular protein orprotein fragments. The sequences of the mRNA (cDNA) and proteins of themarkers of the present invention are known in the art and publiclyavailable.

Alternatively, methylation-sensitive restriction endonucleases can beused to detect methylated CpG dinucleotide motifs. Such endonucleasesmay either preferentially cleave methylated recognition sites relativeto non-methylated recognition sites or preferentially cleavenon-methylated relative to methylated recognition sites. Non limitingexamples of the former are Aat II, Acc III, Ad I, AcI I, Age I, AIu I,Asc I, Ase 1, AsiS I, Ban I, Bbe I, BsaA I, BsaH I, BsiE I, BsiW I, BsrVI, BssK 1, BstB I, BstN I, Bs I, CIa I, Eae I, Eag I, Fau I, Fse I, HhaI, mPl I, HinC II, Hpa 11, Npy99 I, HpyCAIV, Kas I, Mbo I, MIu I, MapA11. Msp I, Nae I, Nar I, Not 1, Pml I, Pst I, Pvu I, Rsr II, Sac II, SapI, Sau3A I, Sfl I, Sfo I, SgrA I, Sma I SnaB I, Tsc I, Xma I, and Zra I.Non limiting examples of the latter are Acc II, Ava I, BssH II, BstU I,Hpa II, Not I, and Mho I.

Alternatively, chemical reagents can be used that selectively modifyeither the methylated or non-methylated form of CpG dinucleotide motifs.Modified products can be detected directly, or after a further reactionwhich creates products that are easily distinguishable. Means whichdetect altered size and/or charge can be used to detect modifiedproducts, including but not limited to electrophoresis, chromatography,and mass spectrometry. Examples of such chemical reagents for selectivemodification include hydrazine and bisulfite ions. Hydrazine-modifiedDNA can be treated with piperidine to cleave it. Bisulfite ion-treatedDNA can be treated with alkali. Other means for detection that arereliant on specific sequences can be used, including but not limited tohybridization, amplification, sequencing, and ligase chain reaction.Combinations of such techniques can be used as is desired.

The principle behind electrophoresis is the separation of nucleic acidsvia their size and charge. Many assays exist for detecting methylationand most rely on determining the presence or absence of a specificnucleic acid product. Gel electrophoresis is commonly used in alaboratory for this purpose.

One may use MALDI mass spectrometry in combination with a methylationdetection assay to observe the size of a nucleic acid product. Theprinciple behind mass spectrometry is the ionizing of nucleic acids andseparating them according to their mass to charge ratio. Similar toelectrophoresis, one can use mass spectrometry to detect a specificnucleic acid that was created in an experiment to determine methylation(Tost, J. et al. 2003).

One form of chromatography, high performance liquid chromatography, isused to separate components of a mixture based on a variety of chemicalinteractions between a substance being analyzed and a chromatographycolumn. DNA is first treated with sodium bisulfite, which converts anunmethylated cytosine to uracil, while methylated cytosine residuesremain unaffected. One may amplify the region containing potentialmethylation sites via PCR and separate the products via denaturing highperformance liquid chromatography (DHPLC). DHPLC has the resolutioncapabilities to distinguish between methylated (containing cytosine) andunmethylated (containing uracil) DNA sequences. Deng, D. et al.describes simultaneous detection of CpG methylation and singlenucleotide polymorphism by denaturing high performance liquidchromatography.

Hybridization is a technique for detecting specific nucleic acidsequences that is based on the annealing of two complementary nucleicacid strands to form a double-stranded molecule. One example of the useof hybridization is a microarray assay to determine the methylationstatus of DNA. After sodium bisulfite treatment of DNA, which convertsan unmethylated cytosine to uracil while methylated cytosine residuesremain unaffected, oligonucleotides complementary to potentialmethylation sites can hybridize to the bisulfite-treated DNA. Theoligonucleotides are designed to be complimentary to either sequencecontaining uracil (thymine) or sequence containing cytosine,representing unmethylated and methylated DNA, respectively.Computer-based microarray technology can determine whicholigonucleotides hybridize with the DNA sequence and one can deduce themethylation status of the DNA. Similarly primers can be designed to becomplimentary to either sequence containing uracil (thymine) or sequencecontaining cytosine. Primers and probes that recognize the convertedmethylated form of DNA are dubbed methylation-specific primers or probes(MSP).

An additional method of determining the results after sodium bisulfitetreatment involves sequencing the DNA to directly observe anybisulfite-modifications. Pyrosequencing technology is a method ofsequencing-by-synthesis in real time. It is based on an indirectbioluminometric assay of the pyrophosphate (PPi) that is released fromeach deoxynucleotide (dNTP) upon DNA-chain elongation. This methodpresents a DNA template-primer complex with a dNTP in the presence of anexonuclease-deficient Klenow DNA polymerase. The four nucleotides aresequentially added to the reaction mix in a predetermined order. If thenucleotide is complementary to the template base and thus incorporated,PPi is released. The PPi and other reagents are used as a substrate in aluciferase reaction producing visible light that is detected by either aluminometer or a charge-coupled device. The light produced isproportional to the number of nucleotides added to the DNA primer andresults in a peak indicating the number and type of nucleotide presentin the form of a program. Pyrosequencing can exploit the sequencedifferences that arise following sodium bisulfite-conversion of DNA.

A variety of amplification techniques may be used in a reaction forcreating distinguishable products. Some of these techniques employ PCR.Other suitable amplification methods include the ligase chain reaction(LCR) (Barringer et al, 1990), transcription amplification (Kwoh et al.1989; WO88/10315), selective amplification of target polynucleotidesequences (U.S. Pat. No. 6,410,276), consensus sequence primedpolymerase chain reaction (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (WO90/06995), nucleic acid based sequenceamplification (NASBA) (U.S. Pat. Nos. 5,409,818; 5,554,517; 6,063,603),microsatellite length polymorphism (MLP), and nick displacementamplification (WO2004/067726).

Sequence variation that reflects the methylation status at CpGdinucleotides in the original genomic DNA offers two approaches to PCRprimer design. In the first approach, the primers do not themselvescover or hybridize to any potential sites of DNA methylation; sequencevariation at sites of differential methylation are located between thetwo primers. Such primers are used in bisulfite genomic sequencing,COBRA, Ms-SNuPE. In the second approach, the primers are designed toanneal specifically with either the methylated or unmethylated versionof the converted sequence. If there is a sufficient region ofcomplementarity, e.g., 12, 15, 18, or 20 nucleotides, to the target,then the primer may also contain additional nucleotide residues that donot interfere with hybridization but may be useful for othermanipulations. Exemplary of such other residues may be sites forrestriction endonuclease cleavage, for ligand binding or for factorbinding or linkers or repeats. The oligonucleotide primers may or maynot be such that they are specific for modified methylated residues.

One way to distinguish between modified and unmodified DNA is tohybridize oligonucleotide primers which specifically bind to one form orthe other of the DNA. After primer hybridization, an amplificationreaction can be performed. The presence of an amplification productindicates that a sample hybridized to the primer. The specificity of theprimer indicates whether the DNA had been modified or not, which in turnindicates whether the DNA had been methylated or not. For example,bisulfite ions convert non-methylated cytosine bases to uracil bases.Uracil bases hybridize to adenine bases under hybridization conditions.Thus an oligonucleotide primer which comprises adenine bases in place ofguanine bases would hybridize to the bisulfite-modified DNA, whereas anoligonucleotide primer containing the guanine bases would hybridize tothe non-converted (initial methylated) cytosine residues in the modifiedDNA. Amplification using a DNA polymerase and a second primer yieldamplification products which can be readily observed. This method isknown as MSP (Methylation Specific PCR; U.S. Pat. Nos. 5,786,146;6,017,704; 6,200,756). Primers are designed to anneal specifically withthe converted sequence representing either the methylated or theunmethylated version of the DNA. Preferred primers and primer sets forassessing the methylation status of the concerned gene by way of MSPwill specifically hybridize to a converted sequence provided in Table 2,or to its complement sequence. Most preferred primers and primer setsare provided in Table 1 and are represented by SEQ ID NO. 1 to 264.Sense primers comprise or consist essentially of SEQ ID NO. 1 to 132,antisense primers consist essentially of SEQ ID NO. 133 to 264. Theamplification products can be optionally hybridized to specificoligonucleotide probes which may also be specific for certain products.Alternatively, oligonucleotide probes can be used which will hybridizeto amplification products from both modified and non-modified DNA.

Thus, present invention provides for a method for identifying cervicalcancer or its precursor, or predisposition to cervical cancer in a testsample containing cervical cells or nucleic acids from cervical cellscomprising: contacting a methylated CpG-containing nucleic acid of atleast one gene selected from the group consisting of genes according toTable 1 with bisulfite to convert unmethylated cytosine to uracil;detecting the methylated CpGs in the nucleic acid by contacting theconverted nucleic acid with oligonucleotide primers whose sequencediscriminates between the bisulfite-treated methylated and unmethylatedversion of the converted nucleic acid; and identifying the test sampleas containing cells that are neoplastic, precursor to neoplastic, orpredisposed to neoplasia, or as containing nucleic acids from cells thatare neoplastic, precursor to neoplastic, or predisposed to neoplasia.

Modified and non-modified DNA can be distinguished with use ofoligonucleotide probes which may also be specific for certain products.Such probes can be hybridized directly to modified DNA or toamplification products of modified DNA. Probes for assessing themethylation status of the concerned gene will specifically hybridize tothe converted sequence but not to the corresponding non convertedsequence. Probes are designed to anneal specifically with the convertedsequence representing either the methylated or unmethylated version ofthe DNA. Preferred converted sequences are provided in Table 2.Preferred probes anneal specifically with the converted sequencerepresenting the methylated version of the DNA, or to the complementsequence thereof. Oligonucleotide probes can be labeled using detectionsystems known in the art. These include but are not limited tofluorescent moieties, radioisotope labeled moieties, bioluminescentmoieties, luminescent moieties, chemiluminescent moieties, enzymes,substrates, receptors, or ligands.

Another way for the identification of methylated CpG dinucleotidesutilizes the ability of the MBD domain of the McCP2 protein toselectively bind to methylated DNA sequences (Cross et al, 1994;Shiraishi et al, 1999). Restriction endonuclease digested genomic DNA isloaded onto expressed His-tagged methyl-CpG binding domain that isimmobilized to a solid matrix and used for preparative columnchromatography to isolate highly methylated DNA sequences. Variants ofthis method have been described and may be used in present methods ofthe invention.

Real time chemistry allows for the detection of PCR amplification duringthe early phases of the reactions, and makes quantitation of DNA and RNAeasier and more precise. A few variants of real-time PCR are well known.They include Taqman® (Roche Molecular Systems), Molecular Beacons®,Amplifluor® (Chemicon International) and Scorpion® DzyNA®, Plexor™(Promega) etc. The TaqMan® system and Molecular Beacon® system haveseparate probes labeled with a fluorophore and a fuorescence quencher.In the Scorpion® system the labeled probe in the form of a hairpinstructure is linked to the primer.

Quantitation in real time format may be on an absolute basis, or it maybe relative to a methylated DNA standard or relative to an unmethylatedDNA standard. The absolute copy number of the methylated marker gene canbe determined; or the methylation status may be determined by using theratio between the signal of the marker under investigation and thesignal of a reference gene with a known methylation (e.g. β-actin), orby using the ratio between the methylated marker and the sum of themethylated and the non-methylated marker.

Real-Time PCR detects the accumulation of amplicon during the reaction,but alternatively end-point PCR fluorescence detection techniques may beused. Confirming the presence of target DNA at the end point stage mayindeed be sufficient and it can use the same approaches as widely usedfor real time PCR.

DNA methylation analysis has been performed successfully with a numberof techniques which are also applicable in present methods of theinvention. These include the MALDI-TOFF, MassARRAY (Ehrich, M. et al.2005), MethyLight (Trinh B. et al. 2001), Quantitative Analysis ofMethylated Alleles (Zeschnigk M. et al. 2004), Enzymatic RegionalMethylation Assay (Galm et al., 2002), HeavyMethyl (Cottrell, S E etal., 2004), QBSUPT, MS-SNuPE (Gonzalgo and Jones, 1997), MethylQuant(Thomassin H. et al. 2004), Quantitative PCR sequencing, andOligonucleotide-based microarray systems (Gitan R S et al., 2006).

The number of genes whose modification is tested and/or detected canvary: one, two, three, four, five, six, seven, eight, nine or more genesaccording to Table 1 can be tested and/or detected. Detection ofepigenetic modification of at least one, two, three, four, five, six,seven, eight, nine or more genes according to Table 1 can be used as anindication of cancer or pre-cancer or risk of developing cancer. Thegenes are preferably selected from the group of JAM3, LMX1A, CDO1, NID2,CCNA1, HOXA11, GREM1 and TAC1. Preferred gene combinations include

-   -   NID2 and HOXA11;    -   JAM3, CDO1, HOXA11, and CCNA1;    -   JAM3 and HOXA11;    -   JAM3, HOXA11 and GREM1;    -   JAM3, NID2, HOXA11 and CDO1;    -   JAM3, TAC1, HOXA11, and CDO1;    -   JAM3, HOXA11, and CDO1;    -   JAM3 and CDO1;    -   JAM3 and NID2;    -   NID2 and CDO1;    -   JAM3 and LMX1A    -   NID2 and LMX1A, and    -   JAM3, CDO1 and NID2.

The accession numbers corresponding to the listed genes can be found atthe website for the National Center for Biotechnology Information. Ofcourse, as appropriate, the skilled person would appreciate thatfunctionally relevant variants of each of the gene sequences may also bedetected according to the methods of the invention. For example, themethylation status of a number of splice variants may be determinedaccording to the methods of the invention. Variant sequences preferablyhave at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% nucleotide sequence identity with the nucleotide sequences in thedatabase entries. Computer programs for determining percentagenucleotide sequence identity are available in the art, including theBasic Local Alignment Search Tool (BLAST) available from the NationalCenter for Biotechnology Information.

It is possible for the methods of the invention to be used in order todetect more than one gene of interest in the same reaction. Through theuse of several specific sets of primers, amplification of severalnucleic acid targets can be performed in the same reaction mixture. Thismay be termed “multiplexing”. Multiplexing can also be utilized in thecontext of detecting both the gene of interest and a reference gene inthe same reaction.

The term “test sample” refers to biological material obtained from amammalian subject, preferably a human subject, and may be any tissuesample, body fluid, body fluid precipitate, or lavage specimen. Testsamples for diagnostic, prognostic, or personalized medicine uses can beobtained from cytological samples, from surgical samples, such asbiopsies, cervical conization or hysterectomy, from (formalin fixed)paraffin embedded cervix or other organ tissues, from frozen tumortissue samples, from fresh tumor tissue samples, from a fresh or frozenbody fluid such as blood, serum, lymph, or from cervical scrapings,cervical smears, cervical washings and vaginal excretions. Such sourcesare not meant to be exhaustive, but rather exemplary. A test sampleobtainable from such specimens or fluids includes detached tumor cellsand/or free nucleic acids that are released from dead or damaged tumorcells. Nucleic acids include RNA, genomic DNA, mitochondrial DNA, singleor double stranded, and protein-associated nucleic acids. Any nucleicacid specimen in purified or non-purified form obtained from suchspecimen cell can be utilized as the starting nucleic acid or acids. Thetest samples may contain cancer cells or pre-cancer cells or nucleicacids from them. Preferably, the test sample contains squamous cellcarcinomas cells or nucleic acids from squamous cell carcinomas,adenocarcinoma cells or nucleic acids of adenocarcinoma cells,adenosquamous carcinoma cells or nucleic acids thereof. Samples maycontain mixtures of different types and stages of cervical cancer cells.

Present invention also relates to screening protocols for the screeningof woman for cervical cancer and the precursors thereof. Traditionallythe Pap Smear has been the primary screening method for the detection ofabnormality of the cervix, but its performance is suboptimal. HumanPapillomavirus has been associated with the development of cervicalcancer. Five high-risk types, 16, 18, 31, 45, and 58, and in particularHPV types 16 and 18 account for approximately 70% of all cervicalcarcinomas. A small percentage of women showing persistent infectionprogress from Low-grade to High-grade lesions. The introduction ofmethylation markers now adds a new dimension to the screening for andtreatment of cervical lesions. Method for cervical cancer screening maycombine high-risk human papillomavirus (hr-HPV) testing and methylationtesting; or cytological evaluation and methylation testing; or hr-HPVtesting and cytological evaluation and methylation testing.

Thus, a further embodiment of the present invention relates to a methodfor cervical cancer detection or screening comprising the steps of:

-   a) providing a test sample comprising cervical cells or nucleic    acids from cervical cells;-   b) assaying the test sample of step a) for high-risk human    papillomavirus (hr-HPV);-   c) if b) is positive for the presence of hr-HPV, assaying the    methylation status of at least one gene selected from the group    consisting of genes according to Table 1;-   d) if the gene of c) is methylated, refer the woman for colposcopy;-   e) if the gene of c) is unmethylated, refer the woman to a more    regular screening for the presence of hr-HPV.

The present invention relates further to a method for cervical cancerdetection or screening comprising the steps of:

-   a) providing a test sample comprising cervical cells or nucleic    acids from cervical cells;-   b) assaying the test sample of step a) for hr-HPV;-   c) if b) is positive for the presence of hr-HPV, assaying the    methylation status of at least one gene selected from the group    consisting of genes according to Table 1, and/or typing the hr-HPV    for the presence of HPV16 and/or HPV18;-   d) if the gene of c) is methylated, and/or HPV16 and/or HPV18    positive, refer the woman for colposcopy;-   e) if the gene of c) is unmethylated, refer the woman to a more    regular screening for the presence of hr-HPV.

In a related embodiment, the invention provides for a method forcervical cancer detection or screening comprising the steps of:

-   a) performing cytology evaluation on a test sample comprising    cervical cells or nucleic acids from cervical cells;-   b) if a) is positive, assaying the methylation status of at least    one gene selected from the group consisting of genes according to    Table 1;-   c) if the at least one gene of b) is methylated, refer the woman for    colposcopy;-   d) if the at least one gene of b) is unmethylated, refer the woman    to cytology testing on a more regular basis.

In a related embodiment, the invention provides for a method forcervical cancer detection or screening comprising the steps of:

-   a) assaying the methylation status of at least one gene selected    from the group consisting of genes according to Table 1;-   b) if the at least one gene of b) is methylated, perform cytology    testing;-   c) if b) is tested positive, refer the woman for colposcopy;-   d) if b) is negative, refer the woman to methylation testing on a    more regular basis.

In all aspects of the invention, the test sample is preferably acervical, cervicovaginal or vaginal sample of a woman.

The phrase “cervical cancer screening” refers to organized periodicprocedures performed on groups of people for the purpose of detectingcervical cancer.

The phrase “assaying for hr-HPV” refers to testing for the presence ofhr-HPV. There are various PCR based assays commercially available tomeasure hr-HPV copy number or viral load in clinical samples. Manytesting methods have been used to detect the presence of HPV incervicovaginal specimens, including viral load quantification, Southernblot, polymerase chain reaction (PCR), ViraPap (Life Technologies,Gaithersburg, Md.), Hybrid Capture tube testing, Hybrid Capturemicrotiter plate assays, and CISH. For instance, assaying for hr-HPV maybe performed with the FDA approved Hybrid Capture II assay (DigeneCorp., Silver Spring, Md.) with a probe cocktail for 13 carcinogenictypes.

The so-called “high risk” HPV types are those strains of HPV more likelyto lead to the development of cancer, while “low-risk” viruses rarelydevelop into cancer. The list of strains considered high risk is beingadapted with the time and the increase in epidemiological knowledge. Assuch, those hr-HPV types comprise, without being limited to, strains 16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 69. Preferred “highrisk” HPV types are HPV16 and HPV18.

The phrase “HPV16 testing” refers to testing for the presence of hr-HPVtype 16. Similarly, “HPV18 testing” refers to testing for the presenceof hr-HPV type 18. The various methods allowing type-specific HPVtesting are well known to the person skilled in the art and areapplicable in the methods of present invention. For instance, testingfor the presence of hr-HPV-16 may be accomplished by PCR amplificationusing primers specific for HPV type 16, which are known by the skilledin the art.

The phrase “performing cytological evaluation” refers to thecytomorphological assessment of cervical samples, which is usuallyperformed by especially trained medical staff. The various methodsallowing cytological testing are well known to the person skilled in theart and are applicable in the methods of present invention. Cytologicalevaluation may be performed with the known Papanicolaou (PAP) smeartest. Alternative means for cytological evaluation include liquid basedcytology with for example the ThinPrep technique (Cytyc Corporation,Marlborough, Mass., USA).

The term “triaging” refers to sorting out or classifying patients inorder to establish priority of treatment's necessity, priority of properplace of treatment, or any other priority in terms of patientmanagement.

The test sample will most of the time be obtained from a subjectsuspected of being tumorigenic or from a subject undergoing routineexamination and not necessarily being suspected of having a disease.Alternatively the sample is obtained from a subject undergoingtreatment, or from patients being checked for recurrence of disease.

Testing can be performed diagnostically or in conjunction with atherapeutic regimen. Testing can be used to monitor efficacy of atherapeutic regimen, whether a chemotherapeutic agent or a biologicalagent, such as a polynucleotide. Epigenetic loss of function of at leastone gene selected from the group consisting of genes according to Table1 can be rescued by the use of DNA demethylating agents and/or DNAmethyltransferase inhibitors. Testing can also be used to determine whattherapeutic or preventive regimen to employ on a patient. Moreover,testing can be used to stratify patients into groups for testing agentsand determining their efficacy on various groups of patients.

Demethylating agents can be contacted with cells in vitro or in vivo forthe purpose of restoring normal gene expression to the cell. Suitabledemethylating agents include, but are not limited to5-aza-2′-deoxycytidine, 5-aza-cytidine, Zebularine, procaine, andL-ethionine. This reaction may be used for diagnosis, for determiningpredisposition, and for determining suitable therapeutic regimes.Accordingly, the invention also provides for a method for predicting thelikelihood of successful treatment or resistance to treatment of cancerwith such agent. If the gene is methylated, the likelihood of successfultreatment is higher than if the gene is unmethylated, or methylated to alesser degree. Conversely, if the gene is unmethylated, or methylated toa lesser degree, the likelihood of resistance to treatment is higherthan if the gene is methylated.

In a related aspect, epigenetic loss of gene function(s) can identifythe stage of the disease and from that the need of treatment.Accordingly, the invention provides for a method for predicting suitabletreatment comprising determining the methylation status of a gene or acombination of genes. If the gene is methylated, the need of cervicalresection is identified; if the gene is unmethylated or methylated to alesser degree, it is decided that there is no need for cervicalresection. In cases of early stage (CIN) and carcinoma in situ, abnormaltissue is removed by cryosurgery, laser surgery, conization, or simplehysterectomy (removal of the uterus). Invasive cervical cancer istreated with radical hysterectomy (removal of the uterus, fallopiantubes, ovaries, adjacent lymph nodes, and part of the vagina).

To attain high rates of tumor detection, it may be necessary to combinethe methods of the invention with established methods and/or markers forcervical cancer identification (Malinowski D, 2007), such asmorphology-based detection methods, HPV methylation testing (Badal etal. 2004, Kalantari et al. 2004), KRAS and BRAF mutation detection (Kanget al. 2007), chromosomal amplification (Rao et al. 2004), proteinexpression (Keating et al. 2001) and HPV detection methods (Brink et al.2007): several HPV detection kits are known in the art and commerciallyavailable, for example kits such as Digene® HPV Test (Qiagen), AMPLICORHPV Test (Roche), HPV High-Risk Molecular Assay (Third WaveTechnologies), LINEAR ARRAY HPV Genotyping Test (Roche), INNO-LiPA HPVGenotyping (Innogenetics), PapilloCheck (Greiner Bio-One GmbH), PreTectHPV-Proofer (Norchip), NucliSENS EasyQ HPV (BioMerieux), F-HPV Typing™(molGENTIX, S.L.) may be utilized. Such examples are not meant to beexhaustive, but rather exemplary.

Another aspect of the invention is a kit for assessing methylation in atest sample. Kits according to the present invention are assemblages ofreagents for testing methylation. They are typically in a package whichcontains all elements, optionally including instructions. The packagemay be divided so that components are not mixed until desired.Components may be in different physical states. For example, somecomponents may be lyophilized and some in aqueous solution. Some may befrozen. Individual components may be separately packaged within the kit.The kit may contain reagents, as described above for differentiallymodifying methylated and non-methylated cytosine residues.

Desirably the kit will contain oligonucleotide primers whichspecifically hybridize to regions within about 10 kbp, within about 5kbp, within about 3 kbp, within about 1 kbp, within about 750 bp, withinabout 500 bp, within 200 bp or within 100 bp kb of the transcriptionstart sites of the genes/markers listed in Table 1.

Typically the kit will contain both a forward and a reverse primer for asingle gene or marker. If there is a sufficient region ofcomplementarity, e.g., 12, 15, 18, or 20 nucleotides, then the primermay also contain additional nucleotide residues that do not interferewith hybridization but may be useful for other manipulations. Exemplaryof such other residues may be sites for restriction endonucleasecleavage, for ligand binding or for factor binding or linkers orrepeats. The oligonucleotide primers may or may not be such that theyare specific for modified methylated residues. The kit may optionallycontain oligonucleotide probes. The probes may be specific for sequencescontaining modified methylated residues or for sequences containingnon-methylated residues. The kit may optionally contain reagents formodifying methylated cytosine residues. The kit may also containcomponents for performing amplification, such as a DNA polymerase anddeoxyribonucleotides. Means of detection may also be provided in thekit, including detectable labels on primers or probes. Kits may alsocontain reagents for detecting gene expression for one of the markers ofthe present invention. Such reagents may include probes, primers, orantibodies, for example. In the case of enzymes or ligands, substratesor binding partners may be sued to assess the presence of the marker.Kits may contain 1, 2, 3, 4, or more of the primers or primer pairs ofthe invention. Kits that contain probes may have them as separatemolecules or covalently linked to a primer for amplifying the region towhich the probes hybridize. Other useful tools for performing themethods of the invention or associated testing, therapy, or calibrationmay also be included in the kits, including buffers, enzymes, gels,plates, detectable labels, vessels, etc.

According to a further aspect, the invention also employs or relies uponor utilizes oligonucleotide primers and/or probes to determine themethylation status of at least one gene selected from a group of genesconsisting of JAM3, LMX1A, CDO1, NID2, ALX3, ALX4, AR, ARID4A, ATM,AURKA, B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf48, C9orf19, CALCA,CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU,COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4,EPB41L3, FOS, FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, GPNMB, GREM1,Gst-Pi, HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1,JPH3, KNDC1, KRAS, LAMA1, LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B,NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC,PRKCE, RAD23B, RALY, RARA, RASSF1A, RBP4, RECK, RPRM, SALL4, SEMA3F,SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST,TAC1, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1,WT1, XRCC3, and ZGPAT. Preferred probes and their sequences bind to atleast one of the polynucleotide sequences listed in Table 2, FIG. 5B orto the complement sequence thereof. Preferred primers and probes areselected from the primers and probes comprising or consistingessentially of the nucleotide sequences set forth in Table 1. Related tothis, the invention also provides for an isolated polynucleotide whichconsists of a nucleotide sequence listed in Table 1, Table 2 and FIG.5B.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

TABLE 1 MSP assays and primer design Official Sense Primer AntisensePrimer Row Gene Gene sequence (5′-3′) sequence (5′-3′) Nr Assay Name IDSymbol Refseq SEQ ID NO's 1-132 SEQ ID NO's 133-264 1 ALX3_25178 257ALX3 NM_006492 GTTTGGTTCGGGTTA CCTACTTATCTCTCCCG GCGT CTCG 2 ALX3_25180257 ALX3 NM_006492 TTGCGTTTTATTTGTA CTTAACGAACGACTTAA TTTCGC CCGACT 3ALX4_25062 60529 ALX4 NM_021926 TTTTATTGCGAGTCGT TATACCGAACTTATCGC CGGTCCTCCG 4 AR_24818 367 AR NM_000044, TGTATAGGAGTCGAA AAACAACTCCGAACGACNM_001011645 GGGACGTA GA 5 ARID4A_24110 5926 ARID4A NM_002892GTTAGGTAAGTGGTA AAAAACGACTACAACTA CGGCGA CGACGA 6 ARID4A_24112 5926ARID4A NM_002892 ATTTAATGAGGACGGT AACAAACTCGCTTCTAC AGGTAGC ACGAA 7ATM_9746 472 ATM XM_940791, TTTAATATAAGTCGGG ATACGACGCAAAAACTANM_900051, TTACGTTCG TCGC NM_138292 8 AURKA_24802 6790 AURKA NR_001587,TTAGGGAGTAAGTGC AAAAACCGATTAACCTA NM_003600 GTTTGC CGCTC 9 B4GALT1_12683 B4GALT1 NM_001497 TAGACGGTTACGAGT CCTTCTTAAAACGACGA AGGCGGTA CGAA10 B4GalT1_3 2683 B4GALT1 NM_001497 TTTTTCGTATTTTAGG TTCCTCCCGAACCTTTAAAGTGGC CGA 11 BMP2_17901 650 BMP2 NM_001200 TTTGGGGTTCGATTATCGAAAACTCCGAAACC ATTTC GAT 12 BMP6_24310 654 BMP6 NM_001718GTTATTTTTCGGCGGG CTAATAATCGCCCCTTC TTC GC 13 BNIP3 664 BNIP3 NM_004052TACGCGTAGGTTTTAA TCCCGAACTAAACGAAA GTCGC CCCCG 14 C13orf18_19885 80183C13orf18 NM_025113 TTTGATTTTTGAAAGC ACACCACGCACCTATAC GTCGT GC 15C13orf18_Gron 80183 C13orf18 NM_025113 TTTTTAGGGAAGTAAAACGTAATACTAAACCCG GCGTCG AACGC 16 C16orf48_22922 84080 C16orf48NM_032140 TAGTTTGGTAGTTAGC AAACCTCCGAAATAACC GGGTC GTC 17 C9orf19_19865152007 C9orf19 NM_022343 ATAGGGGGAGTTCGG ACAATTTACCCCGCTCG TACG ACT 18CALCA_2 796 CALCA NM_001033952 CGTTTTTATAGGGTTT AAATCTCGAAACTCACCNM_001033953 TGGTTGGAC TAACGA NM_001741 19 CAMK4_27356 814 CAMK4NM_001744 TAGTTGTATCGGTTTA CTACCTTCGTACCCTTC GGCGTTT GATT 20 CCNA1_gron8900 CCNA1 NM_003914 GTTATGGCGATGCGG CCAACCTAAAAAACGAC TTTC CGA 21CCND2_25209 894 CCND2 NM_001759 GAAGGTAGCGTTTTTC AAATAAACCCGATCCGC GATGAA 22 CDH1_17968 999 CDH1 NM_004360 AATTTTAGGTTAGAGG ACCAATCAACAACGCGAGTTATCGC AC 23 CDH1_23527 999 CDH1 NM_004360 GAGGGGGTAGGAAAGCGAAACGACCTAAAAAC TCGC CTCG 24 CDH4_24735 1002 CDH4 NM_001794GGGACGATTTTTCGTT TTCTACTACTCTCGCTC GTTC TCCGAC 25 CDK6_9703 1021 CDK6NM_001259 AATTTCGTTTGTAGAG TCTATATTAAAAACTTCG TCGTCGT CTTCG 26CDKN1B_23172 1027 CDKN1B NM_004064 GTCGGTAAGGTTTGG AAAATAACAAAACCCGTAGAGC CCG 27 CDKN2B_27345 1030 CDKN2B NM_004936 TTAGAAGTAATTTAGGAAACCCCGTACAATAAC CGCGTTC CGA 28 CDO1_55928 1036 CDO1 NM_001801AATTTGATTTGTGTGT GAAACGTAAAAATATCG GTATCGC TCGCA 29 CDO1_55929 1036 CDO1NM_001801 GTTTACGCGATTTTTG AAAAACCCTACGAACAC GGAC GACT 30 CLSTN2_1985064084 CLSTN2 NM_022131 AGGGTTTTTCGGAGT TTCCTCAACCGTCTCCA CGTT CG 31CLU_13810 1191 CLU NM_001831, AGGCGTCGTATTTATA TCCCCTTACTTTCCGCGNM_203339 GCGTTT AC 32 CLU_19838 1191 CLU NM_001831, GTGGGGGTCGGTGTATCCCTACTAAAAACGCC NM_203339 GTATC GAA 33 COL1A1_23253 1277 COL1A1NM_000088 TATAAAAGGGGTTCG AAATTAACGTCCGCTCA GGTTAGTC TACG 34 CPT1C_23912126129 CPT1C NM_152359 AGGAAGTATTTATTGC CCATCACTTATCCTCGA GTATGTTTC CGC35 CTDSPL_23795 10217 CTDSPL NM_001008392 TAATTTTAAGGAGGACATAAACTCCAACGACGC GAGGGTC GAAA 36 CTDSPL_23804 10217 CTDSPL NM_005808GTTTTGGGAGAGGCG TCATAATAACGAAACGA GTTC CGACC 37 CYCLIND2_1 894 CCND2NM_001759 GGTGTAGCGTTTAGG CGAATTTTTCCTACGTA (official full GTCGTC ACCGgene name for CCND2) 38 DAPK1 1612 DAPK1 NM_004938 GGATAGTCGGATCGACCCTCCCAAACGCCGA GTTAACGTC 39 DBC1_23879 1620 DBC1 NM_014618AGGATAGGTATGAATT AAACGAACGAACAACAA TCGGTTTC CGA 40 DDX19B_22963 11269DDX19B NM_007242, CGGGTTTGAGGGTAA CGCCACAATAACGTCGA NM_001014449,NM_001015047 TAGAATCG AA 41 DKK2_23970 27123 DKK2 NM_014421GTGCGGGGTAAGAAG AAAAACAATCAAATACG GAAC AAACGC 42 DKK2_23973 27123 DKK2NM_014421 GAGAGAGAAAGCGGG TCACAATTACCCCGAAA AGTTC CG 43 EGFR_23302 1956EGFR NM_201283, TAGGAGCGTTGTTTC CACGACCCCCTAACTCC NM_005228 GGTC GT 44EGR4_24277 1961 EGR4 NM_001965 TTTAGGTGGGAAGCG AAACGCTAAAACCGCGATATTTATC AT 45 EPB41L3_19071 23136 EPB41L3 NM_012307 GGGATAGTGGGGTTGATAAAAATCCCGACGAA ACGC CGA 46 EPB41L3_19072 23136 EPB41L3 NM_012307GCGTGGGTTTTCGTC CCCAAAACTACTCGCCG GTAG CT 47 FOS_22338 2353 FOSNM_005252 CGGGTTGTAGTTAATA CTCTCTCATTCTACGCC TCGAGG GTTC 48 FOXE1_133142304 FOXE1 NM_004473 TTTGTTCGTTTTTCGA TAACGCTATAAAACTCC TTGTTC TACCGC 49GADD45A_24463 1647 GADD45A NM_001924 CGTTAATCGGATAAGA AAAACCACGCGAAAAACGTGCG GA 50 GATA4 2626 GATA4 NM_002052 GTATAGTTTCGTAGTTAACTCGCGACTCGAATC TGCGTTTAGC CCCG 51 GATA4_13295 2626 GATA4 NM_002052GGTATTGTTATTTTGC CCCGAAACAAACTACAC GTTTTC GAC 52 GDAP1L1_19773 78997GDAP1L1 NM_024034 GATTTCGGGTTGTTAT CTAACTTAACCGCATCG GGC CTC 53GDAP1L1_19775 78997 GDAP1L1 NM_024034 GAAAGAAGGAGGTTT CCCGATAAATAATAACACGGC TTCACGA 54 GNB4 59345 GNB4 NM_021629 GTTGTGAGTTGCGTTTCGCTACCGATATCCGCT TTTACGTC AAACG 55 GPNMB_52607 10457 GPNMB NM_001005340GGTCGTAGTCGTAGT CCGCAAAAACCTAAAAC CGGG GTAA 56 GREM1_29777 26585 GREM1NM_013372 GAATTTGGTACGATTT ATCTAAACTTTCCCTAT TACGGAG CGACCG 57Gst-Pi_New3 2950 GSTP1 NM_000852 ATTTAGTATTGGGGCG TAACGAAAACTACGACG GAGCACGA 58 HHIP_23319 64399 HHIP NM_022475 AGTAGTAGGAATAGAAAAAACTACAACCGCCGA ACGGCGA CA 59 HIN1_3 92304 SCGB3A1 NM_052863GAAGTTGGTTAGGGT AACTTCTTATACCCGAT ACGGTC CCTCG 60 HOOK2_19741 29911HOOK2 NM_013312 GGATCGTTGGATTTTG TATATCCTCGCCCCACG GTTC TAA 61HOXA1_27316 3198 HOXA1 NM_153620 TTTTTAGAGTAAATAG ATACGCCTTACAACCCCCGGGAGC TACG 62 HOXA11_23844 3207 HOXA11 NM_005523 TTTTATTTATTCGGGGACAAAATCCTCGTTCTC AGTTGC GAAT 63 HOXA7_2 3204 HOXA7 NM_006896TCGTAGGGTTCGTAG TCCAAATCTTTTTCCGC TCGTTT GA 64 HOXD1(2) 3231 HOXD1NM_024501 GTCGGTTGACGTTTTG ACCGTCTTCTCGAACGA AGATAAGTC CG 65 IGSF4_1898723705 CADM1 NM_014333 TCGGATTTCGTTTTTA GAACACCTACCTCAAAC GCGTAT TAACGAC66 ISYNA1_19726 51477 ISYNA1 NM_016368 TAGGTTGGTTTGGTTTTAAACGACGACCTCCAT CGGTC CG 67 JAM3 83700 JAM3 NM_032801 GGGATTATAAGTCGCCGAACGCAAAACCGAA GTCGC ATCG 68 JPH3_12611 57338 JPH3 NM_020655TTAGATTTCGTAAACG TCTCCTCCGAAAAACGC GTGAAAAC TC 69 KNDC1_19691 85442KNDC1 NM_033404 TGGATGGAGTTTAGG AAAATACTACGAAACCG NM_152643 TTATATCGTCCCC 70 KRAS_24235 3845 KRAS NM_033360 AGGAGGGATTGTCGG GCTCCGAATCAAAATTAATTTAC ACGA 71 LAMA1_63431 284217 LAMA1 NM_005559 TTTTTAGATTTATCGACGAACTCACCTCTCTAC GTGGCG CGAC 72 LMX1A_9513 4009 LMX1A NM_177398,CGGTATCGTTGTTTAG CGTATAACTATTACCTC NM_177399, GAGGC GAAACGCTNM_001033507 73 LOC285016_22940 285016 hCG_1990170 NM_001002919AGTTGTTTGGTATTCG CGACCCCTCCTAACTTT CGGT CG 74 LOX_23395 4015 LOXNM_002317 GTTAGATTGATTTCGT AACTAAAATACCCGTAC TCGAGG TCCGCT 75LTB4R_31250 1241 LTB4R NM_181657 TAGTAGATTTTTAGCG AAAACCTTAACGAAACTGTGAAGACG AAACGAAA 76 MAL 4118 MAL NM_002371 TTCGGGTTTTTTTGTTGAAAACCATAACGACGT TTTAATTC ACTAACG 77 MTAP_24628 4507 MTAP NM_002451GTAAGTGAGTTTCGA CTCCGAAAACCATACGC GTGTCGC CC 78 MYO18B_24620 84700MYO18B NM_032608 GAAAGGTCGGATTTG ACCATCTCATCACGCCT TTTTTC CG 79NDRG2_56603 57447 NDRG2 NM_201540 AGATTTTGTGGTTTCG ATCCCCCGAACATTACGNM_201539 TCGTT ATT NM_201535 NM_201537 80 NID2_9091 22795 NID2NM_007361 GCGGTTTTTAAGGAGT CTACGAAATTCCCTTTA TTTATTTTC CGCT 81NOL4_19645 8715 NOL4 NM_003787 GAGAGATTCGGGATT GTAATCCAAAAATAAAA CGTGACTACGCC 82 NPTX1_2 4884 NPTX1 NM_002522 AGTACGTTGTTTCGGACTTCATCTACACCTCGA GTTTTTC TACCCG 83 NPTX2_57779 4885 NPTX2 NM_002523GCGTCGTTTTGTATGG CCCGATAACCGCTTCGT GTATC AT 84 OGFOD2_23131 79676 OGFOD2NM_024623 CGAGTAGTAGTTGCG ACAAACGACCCTAAAAA TCGGG CGAAC 85 PAK3_1 5063PAK3 NM_002578 TGTATGATTTTAGTTC ACGAATTTTACCTCAAA GCGGAT CGACC 86 PAK3_35063 PAK3 NM_002578 GCGGGATTTATTTGTT AACCCGAAACTACGACT ACGGA ACGAC 87PAX1_27210 5075 PAX1 NM_006192 ATTGCGTCGGGTTTA GCCCCTTACCCATAACG GTTTCAAC 88 PAX1_27211 5075 PAX1 NM_006192 GTTTAGGGAAAGCGG GAACGACAAACAAAACTACGA CGAAA 89 PDCD4_11827 27250 PDCD4 NM_145341, GTTCGTAGTTCGGGGGCGATCCTATCAAATCC NM_014456 CGTT GAA 90 PHACTR3_11692 116154 PHACTR3NM_080672 TTATTTTGCGAGCGGT GAATACTCTAATTCCAC NM_183244 TTC GCGACTNM_183246 91 POMC 5443 POMC NM_000939 GATTTGGGCGTTTTTG GACTTCTCATACCGCAAGTTTTTCGC TCG 92 PRKCE_24134 5581 PRKCE NM_005400 GTGGGTTTTAAGTTTACCTACCCTCGAAACAAA CGGTTTC CGA 93 RAD23B_1 5887 RAD23B NM_002874GGCGGAGTTTGTATA AACCCGAATTACGCAAA GAGGC CG 94 RALY_19607 22913 RALYNM_007367 TTTTTGGGTTTCGTTG CGCCTCAATAATACCGA TTTC CC 95 RARA_24121 5914RARA NM_001024809 TTCGTTTCGTTTAGGT CCTCTCGATTCCCTACG ATCGTTT TTT 96RARA_24129 5914 RARA NM_000964, TTTAGGATTATAGTGA TAACCGCCTTTAACCCCNM_001033603 GCGACGG GA 97 RASSF1A 11186 RASSF1 NM_007182.GCGTTGAAGTCGGGG CCCGTACTTCGCTAACT NM_170712 TTC TTAAACG NM_170714 98RBP4_24106 5950 RBP4 NM_006744 GGTCGTTTCGTTGTTT GCGTTATACAAATACCC TATAGCCCG 99 RECK_18940 8434 RECK NM_021111 TTACGGTTAGTAGAAG CTACGACCAAACTAAATGAGTAGCGT CCGAAC 100 RPRM_2 56475 RPRM NM_019845 TCGAGGAAGAAGATGAAAAACCCGAACGAAC TCGAAG GTAA 101 SALL4_12833 57167 SALL4 NM_020436GAGGCGTAAGTAGGC CGCATCTACAAACTCCG GAAA AAA 102 SEMA3F_23485 6405 SEMA3FNM_004186 GATTAGAGCGAGCGA TAACTACTAAACCCGAA ACGA CCGAAC 103 SLC5A8_24598160728 SLC5A8 NM_145913 GGTTTGTTGGTCGTTT CGAAACATCGACACCTT TTAGC CGT 104SLC5A8_24601 160728 SLC5A8 NM_145913 GTATTTAGGGTAGCG CGAAATAAAAACTAACAGGTCG ATCGCC 105 SLIT1_23651 6585 SLIT1 NM_003061 GCGTTATGGTGTTTTTTCTTCGATAACTCTACC ATAGCGT CCGA 106 SLIT1_23653 6585 SLIT1 NM_003061TTGTAGGCGGTTTGTA GACAATCATCCATCAAT GTCGT CGAAA 107 SLIT2_23672 9353SLIT2 NM_004787 GAGGATCGGTTTAGG CAATTCTAAAAACGCAC TTGC GACT 108SLIT2_23676 9353 SLIT2 NM_004787 AGGGGAAGACGAAGA CACGAACTAACGCTACG GCGTCAA 109 SLIT2_23681 9353 SLIT2 NM_004787 TAGCGGAGAGGAGATGACCCCTACATCTTAAC TACGC AACCG 110 SLIT3_23619 6586 SLIT3 NM_003062AGGGGTATTTATAGG TACCTACTCCGCTACCA CGTTTAGC ACGTAA 111 SMPD1_24061 6609SMPD1 NM_000543 GAAGGGTAATCGGGT CTAATTCGTCTATCCCG GTTTTC TCC 112SOCS1_23595 8651 SOCS1 NM_003745 GATAGGGTTTTGTTTT ATTTTACCCCGCTACCT CGGCCG 113 SOX1_27153 6656 SOX1 NM_005986 TTGTAGTTTTCGAGTT AAAACGATACGCTAAACGGAGGTC CCG 114 SOX1_27159 6656 SOX1 NM_005986 GTTAGGAGTTCGTCGCACCCGAATTACAAATA GTTAGC CCGA 115 SOX17_66072 64321 SOX17 NM_22454GAGATGTTTCGAGGG CCGCAATATCACTAAAC TTGC CGA 116 SPARC_Wis 6678 SPARCNM_003118 TTTCGCGGTTTTTTAG CATACCTCAATAACAAA ATTGTTC CAAACAAACG 117SPN_24052 6693 SPN NM_003123, ATCGTAGGTTGGGTTT AAAAACAAAACACGCGANM_001030288 GGTC AA 118 SST_23808 6750 SST NM_001048 TGGTTGCGTTGTTTATTTACCTACTTCCCCGCG CGTTT AC 119 TAC1_56187 6863 TAC1 NM_003182GGGTATTTATTGCGAC CCGACGACAACTACCG GGAT AAA 120 TERT_23702 7015 TERTNM_003219, GGTTTCGATAGCGTA CTACACCCTAAAAACGC NM_198255 GTTGTTTC GAAC 121TFPI-2 7980 TFPI2 NM_006528 GTTCGTTGGGTAAGG CATAAAACGAACACCCG CGTTCAACCG 122 TLL1_24051 7092 TLL1 NM_012464 TAAGGAATTTTGTATTACCTAACAAACTACGAA CGGAGGC CGCCA 123 TNFAIP1_23212 7126 TNFAIP1 NM_021137GTGGTTAGCGGATTT AACTAAACAACACTCCG CGAGT AACGA 124 TRMT1_19794 55621TRMT1 NM_017722 TTTCGTAGGGTTCGGT CCGAATACTCTCTAAAA GTC CCCGAT 125TWIST1_3 7291 TWIST1 NM_000474 GTTAGGGTTCGGGGG CCGTCGCCTTCCTCCG CGTTGTTACGAA 126 TWIST1_9329 7291 TWIST1 NM_000474 TTTAGTTCGTTAGTTTTACTACTACGCCGCTTA CGTCGGT CGTCC 127 UGT1A1_22912 54658 UGT1A1 NM_007120TTTTGTGGTTAGTCGC ACGTAAAATAAACAATC GGT AACTATCG 128 WIF1_9096 11197 WIF1NM_007191 GCGTCGTTAGATATTT TAACACCCAAACCGAAA TGTTGC AACG 129 WIT1_2456751352 WIT1 NM_015855 GTATGGAGCGTTTTG AACGAATCCACATACCC CGAT GA 130 WT1_17490 WT1 NM_024426, TGTGTTATATCGGTTA CGCTACTCCTTAAAAAC NM_024424GTTGAGAGC GCC 131 XRCC3_9322 7517 XRCC3 NM_005432 CGTTTGTTTTTATAGGACAACGAAATCGAAAAT TTCGGG CGTAA 132 ZGPAT_23961 84619 ZGPAT NM_032527TGTATGCGGAGAGGT ACCATTCCCGACTCCTC NM_181484 CGTAG GT NM_181485

TABLE 2 Amplicon details (converted sequences issuing from themethylated version of the DNA) Official Row Gene Gene Amplicon Sequence(converted) (5′-3′) Nr Assay Name ID Symbol Refseq SEQ ID NO's 265-396 1ALX3_25178 257 ALX3 NM_006492 GTTTGGTTCGGGTTAGCGTTAATTCGGTTTTCGTGGAAGTCGTGGCGAAAGGCGAGAGGGGTAAAAAGTTG AGAAATAGGCGAGCGGGAGAGATAAGTAGG 2ALX3_25180 257 ALX3 NM_006492 TTGCGTTTTATTTGTATTTCGCGTCGTTTCGCGGTTCGCGGTTGATTCGTTTTTCGGTTTGCGGGTTTTTGGAGTTTTATTTTTTAGAGTCGGTTAAGTCGTTCGTTAAG 3 ALX4_25062 60529 ALX4 NM_021926TTTTATTGCGAGTCGTCGGTCGTTGTTATGGACGTTTATTATAGTTCGGTGTCGTAGAGTCGGGAGGGTTCGTCGTTTTTTAGGGTATTTTTCGGAGGCGATAAGTTCG GTATA 4 AR_24818 367 AR NM_000044,TGTATAGGAGTCGAAGGGACGTATTACGTTAGTTTT NM_001011645AGTTCGGTTTTAGCGATAGTTAACGTTTTTTGTAGCG CGGCGGTTTCGAAGTCGTCGTTCGGAGTTGTTT5 ARID4A_24110 5926 ARID4A NM_002892 GTTAGGTAAGTGGTACGGCGAGCGTAAGGGAAGGGGTTAGTTATTGATTAGCGGTAGTAATTGTAGGAATCG TCGTCGTAGTTGTAGTCGTTTTT 6ARID4A_24112 5926 ARID4A NM_002892 ATTTAATGAGGACGGTAGGTAGCGAGGTTTTATTCGAAGTTTTTCGGCGTTATGAGTAGTTAATAGGAGTTC GTGTAGAAGCGAGTTTGTT 7 ATM_9746 472ATM XM_940791, TTTAATATAAGTCGGGTTACGTTCGAGGGTAATAATA NM_000051,TGATTAAAATTATAGTAGGAATTATAATAAGGAATAA NM_138292GATTTAGGTTAAAGTAAATATAGCGATAGTTTTTGCG TCGTAT 8 AURKA_24802 6790 AURKANR_001587, TTAGGGAGTAAGTGCGTTTGCGCGCGGTGTGCGTT NM_003600TTTAAACGCGATTTAAGGCGTCGGGTTTGTTGTTAATTAATTATAAGGTAGTTTCGTTCGAGCGTAGGTTAATC GGTTTTT 9 B4GALT1_1 2683 B4GALT1NM_001497 TAGACGGTTACGAGTAGGCGGTAGGTTCGTTGTAGGGACGCGTTTGGTATCGCGGCGTTGTCGTTTAGGAGCGGTTTTCGAAGTTTTATTTTTTCGTCGTCGTTTTA AGAAGG 10 B4GalT1_3 2683 B4GALT1NM_001497 TTTTTCGTATTTTAGGAAGTGGCGCGGTTTGTCGAGGGTAGCGTGGAGGAGGAAGAGGAGGCGCGGTTTAA CGCGATCGAAGTTTCGTCGTAAAGGTTCGGGAGGAA11 BMP2_17901 650 BMP2 NM_001200 TTTGGGGTTCGATTATATTTCGGTTAGCGCGTTTTAGGTTTTCGATTTTTTGTAGTAGGTGTTTCGTATCGCGG CGTTAGGGATCGGTTTCGGAGTTTTCG 12BMP6_24310 654 BMP6 NM_001718 GTTATTTTTCGGCGGGTTCGTTTTTTTTTTTTGGTTTTTAGTTTTTATTTTTTATGGTCGTTCGGGGCGTTTTTAGTTGTTTAGGTTAGAGAGGTGGCGAAGGGGCGATT ATTAG 13 BNIP3 664 BNIP3 NM_004052TACGCGTAGGTTTTAAGTCGCGGTTAATGGGCGACGCGGTCGTAGATTCGTTCGGTTTCGTTTTGTTTTGTGAGTTTTTTCGGTCGGGTTGCGGGGTTTCGTTTAGTTC GGGA 14 C13orf18_19885 80183C13orf18 NM_025113 TTTGATTTTTGAAAGCGTCGTTGCGTTTCGCGTCGCGGGTAGGTAGGGCGGGATTTTTAGGAGGATCGGTA GAGGCGCGTATAGGTGCGTGGTGT 15C13orf18_Gron 80183 C13orf18 NM_025113TTTTTAGGGAAGTAAAGCGTCGTTTTCGTCGTAGGTATCGAGACGTCGTTTAGATGGAAGAAATTTTGGAGATGCGCGTTTTTATATCGGTGTCGCGGCGTTCGGGTT TAGTATTACGT 16 C16orf48_22922 84080C16orf48 NM_032140 TAGTTTGGTAGTTAGCGGGTCGGGGCGTTTAGTTTTATTTTTTAGAGCGTTGCGGTTTTGTGTTTGAAGGTTA AATAGTTTGACGGTTATTTCGGAGGTTT 17C9orf19_19865 152007 C9orf19 NM_022343ATAGGGGGAGTTCGGTACGGCGCGGGCGTTTAGGA GAGAAGGAATAATAAATGGATGAGGGGGATGTTTAGGGTTGTTTTCGGGATAGTCGAGCGGGGTAAATTGT 18 CALCA_2 796 CALCA NM_001033952CGTTTTTATAGGGTTTTGGTTGGACGTCGTCGTCGT NM_001033953CGTTGTTATCGTTTTTGATTTAAGTTATTTTTCGTTAG NM_001741 GTGAGTTTCGAGATTT 19CAMK4_27356 814 CAMK4 NM_001744 TAGTTGTATCGGTTTAGGCGTTTTGGTGGGGTGGGAAGGATTCGAGTCGTATTTGAATGAAGGTTAGTTTTTTTTTAAGATATTAATTAGGTAGGGAGAAATCGAAGGG TACGAAGGTAG 20 CCNA1_gron 8900CCNA1 NM_003914 GTTATGGCGATGCGGTTTCGGAGAGCGTACGTTTGTCGCGGTCGGTATGGAAACGTTTTCGTTAGGTTCGG GGGCGTCGTTGATTGGTCGATTTAATAGACGCGGGTGGGTAGTTTAGTCGTATCGTTAAGTTCGGTCGTTTTT TAGGTTGG 21 CCND2_25209 894 CCND2NM_001759 GAAGGTAGCGTTTTTCGATGGTGAGTAGGTTTTGTAGGACGCGGTCGTTTCGGAGTAGGTTGCGGTTTCGT ACGGTTTTGCGGATCGGGTTTATTT 22CDH1_17968 999 CDH1 NM_004360 AATTTTAGGTTAGAGGGTTATCGCGTTTATGCGAGGTCGGGTGGGCGGGTCGTTAGTTTCGTTTTGGGGAG GGGTTCGCGTTGTTGATTGGT 23 CDH1_23527999 CDH1 NM_004360 GAGGGGGTAGGAAAGTCGCGTTCGTTTTTTATTATTTATTTTTTATTTTTATTATTGGGGGGTTCGGAGCGCG CGAGGTTTTTAGGTCGTTTCG 24CDH4_24735 1002 CDH4 NM_001794 GGGACGATTTTTCGTTGTTCGGGGTTTTCGAACGGCGGGGGCGGGAGGCGGTAATTTATTCGGAGCGCGTC GGAGAGCGAGAGTAGTAGAA 25 CDK6_97031021 CDK6 NM_001259 AATTTCGTTTGTAGAGTCGTCGTCGTCGTCGTCGTCGGAGGAGCGAGTCGATTTTTTTTTTTTTTTTTTCGAA GCGAAGTTTTTAATATAGA 26CDKN1B_23172 1027 CDKN1B NM_004064 GTCGGTAAGGTTTGGAGAGCGGTTGGGTTCGCGGGATTCGCGGGTTTGTATTCGTTTAGATTCGGACGGGT TTTGTTATTTT 27 CDKN2B_27345 1030CDKN2B NM_004936 TTAGAAGTAATTTAGGCGCGTTCGTTGGTTTTTGAGCGTTAGGAAAAGTTCGGAGTTAACGATCGGTCGTTC GGTTATTGTACGGGGTTT 28 CDO1_559281036 CDO1 NM_001801 AATTTGATTTGTGTGTGTATCGCGTTTTTAGCGATTTCGGATTTATTGCGTTGTTAGGGGTTTGGGGGTGGGT TTTTTGTTGTTTTTGCGACGATATTTTTACGTTTC29 CDO1_55929 1036 CDO1 NM_001801 GTTTACGCGATTTTTGGGACGTCGGAGATAACGGGGTTTTTGGGAAGGCGCGGAGTTCGGGGAAGTCGGG GATGTGCGCGTGAGTCGTGTTCGTAGGGTTTTT 30CLSTN2_19850 64084 CLSTN2 NM_022131 AGGGTTTTTCGGAGTCGTTTATTAGGGTTTTTTGGGGGTTCGGTTTCGATTGGGTAGGGGGATTTGGATAG GGTTTCGGAGCGTGGAGACGGTTGAGGAA 31CLU_13810 1191 CLU NM_001831, AGGCGTCGTATTTATAGCGTTTTGTTCGCGTATATATNM_203339 TTTTTTTGGGGTTGGTTGTAAATTTGTATGATTTACGTTTAAAGAATGTCGCGGAAAGTAAGGGGA 32 CLU_19838 1191 CLU NM_001831,GTGGGGGTCGGTGTAGTATCGGGTTGGGGGCGTC NM_203339GGGGGGCGTATTATTATTACGAATAGTTGTGTTGGTTTTAGGAGAGATTTTGAGGTGCGGTCGTTCGGCGTT TTTAGTAGGGA 33 COL1A1_23253 1277COL1A1 NM_000088 TATAAAAGGGGTTCGGGTTAGTCGTCGGAGTAGACGGGAGTTTTTTTTCGGGGTCGGAGTAGGAGGTACG CGGAGTGTGAGGTTACGTATGAGCGGACGTTAATTT34 CPT1C_23912 126129 CPT1C NM_152359AGGAAGTATTTATTGCGTATGTTTCGTAGTTTGGGATGTTGAGGTTGTGAGCGGAGGCGAGCGTCGAGGATA AGTGATGG 35 CTDSPL_23795 10217CTDSPL NM_001008392 TAATTTTAAGGAGGACGAGGGTCGGTTGTCGGGCGCGGGCGAGAAAGGTGAGGAGGGGCGTAGGCGGTC GCGGGTTGGGGGCGAGCGTATATTTCGCGTCGTTGGAGTTTAT 36 CTDSPL_23804 10217 CTDSPL NM_005808GTTTTGGGAGAGGCGGTTCGGGTTCGCGTTTTAGTT TTCGTCGTCGTCGTCGTTGGGTTCGAGCGGTCGTCGTTTCGTTATTATGA 37 CYCLIND2_1 894 CCND2 NM_001759GGTGTAGCGTTTAGGGTCGTCGTAGGTCGGGGGTA (official full geneGGGTTTTTAGCGGTTTTTTCGCGGTTAGCGGTTACG name for CCND2) TAGGAAAAATTCG 38DAPK1 1612 DAPK1 NM_004938 GGATAGTCGGATCGAGTTAACGTCGGGGATTTTGTTTTTTTCGCGGAGGGGATTCGGTAATTCGTAGCGGTA GGGTTTGGGGTCGGCGTTTGGGAGGG 39DBC1_23879 1620 DBC1 NM_014618 AGGATAGGTATGAATTTCGGTTTCGGAAGGCGGTTATTATTTTTTTTGTTTTTCGGTTTTTTCGTTTTCGTTTTC GTTGTTGTTCGTTCGTTT 40DDX19B_22963 11269 DDX19B NM_007242,CGGGTTTGAGGGTAATAGAATCGATAGTTTTAAGTG NM_001014449,GGTAAAGGGTGGTTAAATAGGAGTGGTTTTCGACGT NM_001015047 TATTGTGGCG 41DKK2_23970 27123 DKK2 NM_014421 GTGCGGGGTAAGAAGGAACGGAAGCGGTGCGATTTATAGGGTTGGGTTTTTTTGTATTTTGGGTTACGTTTTTTTGGCGAGAAAGCGTTTCGTATTTGATTGTTTTT 42 DKK2_23973 27123 DKK2 NM_014421GAGAGAGAAAGCGGGAGTTCGCGGCGAGCGTAGC GTAAGTTCGTTTTTTAGGTATCGTTGCGTTGGTAGCGATTCGTTGTTTTTTGTGAGTTAGGGGATAACGTTTC GGGGTAATTGTGA 43 EGFR_23302 1956EGFR NM_201283, TAGGAGCGTTGTTTCGGTCGTTTCGGAGGGTCGTAT NM_005228CGTTGTTTTTCGAAGAGTTCGTTTCGGTTTTTTCGAT TAATATTGGACGGAGTTAGGGGGTCGTG 44EGR4_24277 1961 EGR4 NM_001965 TTTAGGTGGGAAGCGTATTTATCGGACGGTCGGTTCGGTGAGGCGTAGCGTTTTAGATTGGCGTATTCGCG GTTTTAGCGTTT 45 EPB41L3_19071 23136EPB41L3 NM_012307 GGGATAGTGGGGTTGACGCGTGGTTTCGGCGTCGCGCGGTTTTTCGAATTTCGAGTTTCGCGTTCGGCGCGGTCGGGGTTTTTAATCGTTTTTTCGTTCGTCGGGATT TTTAT 46 EPB41L3_19072 23136EPB41L3 NM_012307 GCGTGGGTTTTCGTCGTAGTTTCGCGGAGTTTCGGTGTTTTTTGTAATAGGGGGCGGGGGGAATAGCGGCG AGTAGTTTTGGG 47 FOS_22338 2353 FOSNM_005252 CGGGTTGTAGTTAATATCGAGGGTGTAGTGCGGGGGGAGGCGGGGGTCGCGGTTGGGGGAGGGGAGGC GGGAACGGCGTAGAATGAGAGAG 48 FOXE1_133142304 FOXE1 NM_004473 TTTGTTCGTTTTTCGATTGTTCGTTTTTCGGGGTTCGGGCGTATTTTTTTAGGTAGGAGTAGTTGTGGCGGCG CGGTAGGAGTTTTATAGCGTTA 49GADD45A_24463 1647 GADD45A NM_001924CGTTAATCGGATAAGAGTGCGCGCGGGATTCGTTTTTTTTTTTCGGTATCGTTTTCGTTTTCGTTTTTTCGGTT CGTTTTTCGCGTGGTTTT 50 GATA4 2626GATA4 NM_002052 GTATAGTTTCGTAGTTTGCGTTTAGCGGAGGTGTAGTCGGGGTCGCGTATTTTCGTTTCGTTTTTGTACGTGATTTTTATAGGTTAGTTAGCGTTTTAGGGTCGAGTTG TTGGGTCGGGGATTCGAGTCGCGAGTT 51GATA4_13295 2626 GATA4 NM_002052 GGTATTGTTATTTTGCGTTTTCGGAGTCGTTGGTGGGCGATAAGTTTTCGTTTATTTTTTTTTATGTGCGAGTT GTCGTGTAGTTTGTTTCGGG 52GDAP1L1_19773 78997 GDAP1L1 NM_024034GATTTCGGGTTGTTATGGCGATTTTTAATAATTTGATTTTTATTAATTGTAGTTGGTGGTTTATTTTCGCGTTG GAGAGCGATGCGGTTAAGTTAG 53GDAP1L1_19775 78997 GDAP1L1 NM_024034GAAAGAAGGAGGTTTCGGCGCGGCGGTTTTTTTTCGTTTAGTATTATATGGTTTCGTCGAGTTTGTTTTTTTTTTTTTTTTTTTTTCGTTTCGTGAATGTTATTATTTATCG GG 54 GNB4 59345 GNB4 NM_021629GTTGTGAGTTGCGTTTTTTACGTCGGTTTCGCGTTTTAGGGGTTGTTGAGCGTTTAGCGGATATCGGTAGCG 55 GPNMB_52607 10457 GPNMBNM_001005340 GGTCGTAGTCGTAGTCGGGAGATTGAGGGTTAGGGCGCGGTCGCGGGGTTTTTTGGGTCGGGGCGCGGTT TACGTTTTAGGTTTTTGCGG 56 GREM1_2977726585 GREM1 NM_013372 GAATTTGGTACGATTTTACGGAGATTTCGTTTTTTTTAGCGTAGTTTTCGTTATTGAGCGCGGGATTAACGTA GGCGATGTCGGGCGGTCGATAGGGAAAGTTTAGAT57 Gst-Pi_New3 2950 GSTP1 NM_000852 ATTTAGTATTGGGGCGGAGCGGGGCGGGATTATTTTTATAAGGTTCGGAGGTCGCGAGGTTTTCGTTGGAGT TTCGTCGTCGTAGTTTTCGTTA 58HHIP_23319 64399 HHIP NM_022475 AGTAGTAGGAATAGAAACGGCGACGGCGGCGGCGGGGTAGGCGGAGGTAGGGTTAGCGTTGGGTTTTAGATGATGTTGAGGTTTTTTTTGTCGGCGGTTGTAGTTTT 59 HIN1_3 92304 SCGB3A1 NM_052863GAAGTTGGTTAGGGTACGGTCGTGAGCGGAGCGGG TAGGGTTTTTTTAGGAGCGCGGGCGAGGTCGGCGTTGGAGGGGCGAGGATCGGGTATAAGAAGTT 60 HOOK2_19741 29911 HOOK2 NM_013312GGATCGTTGGATTTTGGTTCGAGTATTCGTTTTCGTTACGTGGTAAGTTTGCGTGGAAAGGATAGGTGAGGTTTCGTTTTTTTGTGGTTGGTTTACGTGGGGCGAGGAT ATA 61 HOXA1_27316 3198 HOXA1NM_153620 TTTTTAGAGTAAATAGCGGGAGCGTATTGGGGGTATTTATTATTTACGTTTGTTTTTTGATTTAACGCGTAGG GGTTGTAAGGCGTAT 62 HOXA11_238443207 HOXA11 NM_005523 TTTTATTTATTCGGGGAGTTGCGGGTGGGAGGTGGGGACGAGAGTTGAGTTTTTATCGTTTTTTGTATATTC GAGAACGAGGATTTTGT 63 HOXA7_2 3204HOXA7 NM_006896 TCGTAGGGTTCGTAGTCGTTTAGAATGGAAGGGTAAGAGGTTTAAATATGCGGTTAAAGAATTCGTTCGCGT TCGGCGGGTTTGGCGCGTTTCGCGGAAAAAGATTTGGA 64 HOXD1(2) 3231 HOXD1 NM_024501GTCGGTTGACGTTTTGAGATAAGTCGGAAAAGGGTCGGGTTCGTCGAAGGTCGCGTAATTTATTTGGTCGTT GAGGAGGAAAGAGTCGTCGTTCGAGAAGACGGT65 IGSF4_18987 23705 CADM1 NM_014333TCGGATTTCGTTTTTAGCGTATGTTATTAGTATTTTATTAGTTGTTCGTTCGGGTTTCGGAGGTAGTTAACGTC GTTAGTTTGAGGTAGGTGTTC 66ISYNA1_19726 51477 ISYNA1 NM_016368TAGGTTGGTTTGGTTTCGGTCGTTTAGAGTTTTCGTTGATTTTTTGTTTATTTCGGGTTTTTAGTTCGTCGCGA TGGAGGTCGTCGTTTA 67 JAM3 83700JAM3 NM_032801 GGGATTATAAGTCGCGTCGCGTTGTCGTTGGTTTTTTAGTAATTTTCGATATGGCGTTGAGGCGGTTATCGC GATTTCGGTTTTGCGTTCG 68 JPH3_1261157338 JPH3 NM_020655 TTAGATTTCGTAAACGGTGAAAACGGATTTAGGCGATCGATATAGTAGAGTCGCGGTCGTCGGCGGTTTTG GGTCGCGAGCGTTTTTCGGAGGAGA 69KNDC1_19691 85442 KNDC1 NM_033404 TGGATGGAGTTTAGGTTATATCGTCGAGTTGTTTGTNM_152643 GCGTGTTATTTTTGGAAGTTATTTCGTGTGTTAATTA TGTAGGGCGGTTTCGTAGTATTTT70 KRAS_24235 3845 KRAS NM_033360 AGGAGGGATTGTCGGATTTACGCGGCGGTTCGTTTTTTGTTTAGTCGTAAGGTTGTTTTCGTAGTCGTTAATT TTGATTCGGAGC 71 LAMA1_63431 284217LAMA1 NM_005559 TTTTTAGATTTATCGAGTGGCGGCGGAGGCGAGATGCGCGGGGGCGTGTTTTTGGTTTTGTTGTTGTGTGTC GTCGCGTAGTGTCGGTAGAGAGGTGAGTTCG 72LMX1A_9513 4009 LMX1A NM_177398, CGGTATCGTTGTTTAGGAGGCGTCGATATTTTCGTANM_177399, AAGGTTTAGTCGGGGTGAGGGGTATTGGGGGGCGA NM_001033507TCGGGTTAGAGCGTTTCGAGGTAATAGTTATACG 73 LOC285016_22940 285016 hCG_1990170NM_001002919 AGTTGTTTGGTATTCGCGGTTTTTAAAGGGGAAAGAAAGTTGCGTTCGCGTTAGGCGTAGCGCGTTCGGCG GACGCGGTTTTTCGGGCGAAAGTTAGGAGGGGTCG74 LOX_23395 4015 LOX NM_002317 GTTAGATTGATTTCGTTCGAGGAGGACGTGGTTTATAGAAAATAAAAACGGGGTTTAAATTACGTGAGGGAAGGAGAAATTTTTAATTAAGGAGGCGAGCGGAGTACG GGTATTTTAGTT 75 LTB4R_31250 1241LTB4R NM_181657 TAGTAGATTTTTAGCGGTGAAGACGTAGAGTATCGGGTTGACGTTAGAATTGAAGAAGGTTAAGGTCGTAGTTTTCGTTCGCGTCGTTTGGTCGGTTTCGTTTAGTTTC GTTAAGGTTTT 76 MAL 4118 MALNM_002371 TTCGGGTTTTTTTGTTTTTAATTCGCGCGCGGGGGCGTTTAGGTTATTGGGTTTCGCGGAGTTAGCGAGAGGTTTGCGCGGAGTTTGAGCGGCGTTCGTTTCGTTTTA AGGTCGACGTTAGTACGTCGTTATGGTTTTC 77MTAP_24628 4507 MTAP NM_002451 GTAAGTGAGTTTCGAGTGTCGCGTTTTAGTTTTTTTTCGCGGCGGTAAGGGACGTACGGGTCGGGCGTATG GTTTTCGGAG 78 MYO18B_24620 84700MYO18B NM_032608 GAAAGGTCGGATTTGTTTTTCGAGGGTCGAGTTAGTTTTTGTAGATGGTTGTAGTTTTAGTTATGAGTGTTATTTTTTTTTTGTTTTTATAGGGCGAGGCGTGATGAGAT GGT 79 NDRG2_56603 57447 NDRG2NM_201540, NM_201539, AGATTTTGTGGTTTCGTCGTTAATTTTTTTTAGTTCG NM_201535,NM_201537 GTTTAGAATAGGAGATTAGTTTAGGTTCGTTGAATCG TAATGTTCGGGGGAT 80NID2_9091 22795 NID2 NM_007361 GCGGTTTTTAAGGAGTTTTATTTTCGGGATTAAATGGTTCGTAAGGTTTGGGGTAGCGGCGTTGTAGGAGAT GAGTTTAGCGTAAAGGGAATTTCGTAG 81NOL4_19645 8715 NOL4 NM_003787 GAGAGATTCGGGATTCGTGTGTTTTTCGGGGTTTAAAGGCGTTGGGCGGGCGGTTGTTTTCGGGAGAGGC GTAGTTTTTATTTTTGGATTAC 82 NPTX1_24884 NPTX1 NM_002522 AGTACGTTGTTTCGGAGTTTTTCGGCGTCGTCGGCGGTTACGGACGCGGCGTATATGTCGGCGTTTACGGG TATCGAGGTGTAGATGAAG 83 NPTX2_577794885 NPTX2 NM_002523 GCGTCGTTTTGTATGGGTATCGCGGGTAGCGGGTAGTCGGCGTGTATCGTTTTTGGGGGTAGTGTCGTGTA TACGAAGCGGTTATCGGG 84 OGFOD2_2313179676 OGFOD2 NM_024623 CGAGTAGTAGTTGCGTCGGGATTACGGTTCGGTGAGTGGTCGTTGTCGTTTTTACGGAGTAGTGGGTAGAG AGGGGTAGTGGAGGAGGGAAGTTCGTTTTTAGGGTCGTTTGT 85 PAK3_1 5063 PAK3 NM_002578TGTATGATTTTAGTTCGCGGATAAGTGGGTGTGTTAGGGTCGTTTTTAGAGGGTCGGGGTTTTTTCGTTTGGTTAAATTTTAGATTCGTTTATTGGGGTTTGGGTCGTT TGAGGTAAAATTCGT 86 PAK3_3 5063PAK3 NM_002578 GCGGGATTTATTTGTTACGGATTTAGTTATTTCGTTAAGATTTTTTTTTTATTTTCGAGCGTTTTAGTTGGCGGGGTTGGGGAGTCGTAGTTTCGCGGTCGTAGTCGTA GTTTCGGGTT 87 PAX1_27210 5075 PAX1NM_006192 ATTGCGTCGGGTTTAGTTTCGGTTATTTCGGTTATTTCGGCGTTAGGTAGTTGGTCGGTTCGTTCGTTATGGG TAAGGGGC 88 PAX1_27211 5075 PAX1NM_006192 GTTTAGGGAAAGCGGACGAGAGGGAAGGGAGGTAGGCGGATTCGATTTATTTTATTAGTTTTTTCGAGTTTT GTTTGTCGTTC 89 PDCD4_11827 27250PDCD4 NM_145341, GTTCGTAGTTCGGGGCGTTGGGGAGGGCGCGGTTG NM_014456GATTTGCGGGGTTATAAGAAGGTAGTCGGATTTTCG TATCGTAGGTTCGGATTTGATAGGATCGC 90PHACTR3_11692 116154 PHACTR3 NM_080672TTATTTTGCGAGCGGTTTCGCGATACGAGGTAGTCG NM_183244TTTTCGTTTTTCGACGCGGTTATGGGTTCGGTCGGC NM_183246GCGGGGGTAAGTTAGAGCGAGTCGCGTGGAATTAG AGTATTC 91 POMC 5443 POMC NM_000939GATTTGGGCGTTTTTGGTTTTTCGCGGTTTCGAGTTTTCGATAAATTTTTTGCGTCGATTGCGGTATGAGAAGTC 92 PRKCE_24134 5581 PRKCENM_005400 GTGGGTTTTAAGTTTACGGTTTCGTAGATTTTGATTTTAAGAAGGTTATTGAATATTATTATGGTCGGGGCGG GGAGTGGGGGTCGGGGTTATTTCGTTTGTTTCGAGGGTAGG 93 RAD23B_1 5887 RAD23B NM_002874GGCGGAGTTTGTATAGAGGCGGAGTCGCGGTAGTC GGAGAGAACGTTTTAGTAATAGTCGTTAGGAGGAAGTTTTAGGAGTTTTTGTCGTTTACGGAACGCGTTTGC GTAATTCGGGTT 94 RALY_19607 22913RALY NM_007367 TTTTTGGGTTTCGTTGTTTCGAGTTGGCGTCGTTCGCGCGTTTCGTCGTATTGATAGCGGCGCGAGTTTCGTAATCGCGAGTTTTGTTTTCGGTCGGTATTATTGAGG CG 95 RARA_24121 5914 RARANM_001024809 TTCGTTTCGTTTAGGTATCGTTTTTGGTTTAATTTATTTTCGGCGCGTTCGGTTGTAGCGGGAGAAACGTAGG GAATCGAGAGG 96 RARA_24129 5914 RARANM_000964, TTTAGGATTATAGTGAGCGACGGGAGAGGAGGGAT NM_001033603GGGGAAAGTTAGAATTGGCGAGAAGGAAATGGTTA GATTAGAAGTAGAGGTCGGGGTTAAAGGCGGTTA97 RASSF1A 11186 RASSF1 NM_007182 GCGTTGAAGTCGGGGTTCGTTTTGTGGTTTCGTTCGNM_170712 GTTCGCGTTTGTTAGCGTTTAAAGTTAGCGAAGTAC NM_170714 GGG 98RBP4_24106 5950 RBP4 NM_006744 GGTCGTTTCGTTGTTTTATAGCGTCGGGGGGAGGGGGTCGCGTTTTCGTAATCGCGCGGGGTGAAAGATC GAAGGGGAGGCGTCGGGGGTATTTGTATAACGC 99RECK_18940 8434 RECK NM_021111 TTACGGTTAGTAGAAGGAGTAGCGTATTTCGTAGAGAGGTTCGGACGGTCGTTATGTTCGGGTCGGGCGGT TTTAGAGTCGCGGGATGTTCGGATTTAGTTTGGTCGTAG 100 RPRM_2 56475 RPRM NM_019845 TCGAGGAAGAAGATGTCGAAGATTACGGTGAGTGAGAGTACGTATATGATCGCGATTTGTATTACGCGTATT ATGTATAGGTTACGTTCGTTCGGGTTTTT 101SALL4_12833 57167 SALL4 NM_020436 GAGGCGTAAGTAGGCGAAATTTTAGTATATTAATTCGGAGGAGGATTAGGGCGAGTAGTAGTCGTAGTAGT AGATTTCGGAGTTTGTAGATGCG 102SEMA3F_23485 6405 SEMA3F NM_004186 GATTAGAGCGAGCGAACGAATCGCGGCGGTTCGGAGAGTTTCGAGCGTAGCGTAGGATTTGGGTACGTCG CGAGGAATCGTGTAGTTTAGCGCGGTCGTTCGGTTCGGGTTTAGTAGTTA 103 SLC5A8_24598 160728 SLC5A8 NM_145913GGTTTGTTGGTCGTTTTTAGCGAAGGCGTAGTAGATGTCGATGGCGGTCGAGATGATTAGTATGTTCGCGAATATTACGTAGTTTTATATTACGAAGGTGTCGATGTTT CG 104 SLC5A8_24601 160728 SLC5A8NM_145913 GTATTTAGGGTAGCGGGTCGATTTTTCGAGGTTTTATATTTGGGTTTGAGGGGCGCGGTTCGTAGCGGCGG GTGTAGGGGCGATTGTTAGTTTTTATTTCG 105SLIT1_23651 6585 SLIT1 NM_003061 GCGTTATGGTGTTTTTATAGCGTTTCGTTCGCGAGTTAGACGGTAGTAGTCGTTGATTATTTTCGTTCGGGGTCGTTTTTAGGTGTAGTTTCGGGGTAGAGTTATCGAA GA 106 SLIT1_23653 6585 SLIT1NM_003061 TTGTAGGCGGTTTGTAGTCGTTGAGTGGTCGTCGGGAGAGGGGGGTTGCGGCGGGGGAGGGCGGGGAG GAGTTTGGTTTTGGATGTGTGTTTTTCGATTGATGGATGATTGTC 107 SLIT2_23672 9353 SLIT2 NM_004787GAGGATCGGTTTAGGTTGCGGCGGAGTCGAGGGCG AGGGAGAGGTCGCGTGAGTGAGTAGAGTTTAGAGTCGTGCGTTTTTAGAATTG 108 SLIT2_23676 9353 SLIT2 NM_004787AGGGGAAGACGAAGAGCGTATATTTATAGTTTTTCGGTGTTGCGGGGGATATTTTTGGGTACGTTGCGTAGC GTTAGTTCGTG 109 SLIT2_23681 9353SLIT2 NM_004787 TAGCGGAGAGGAGATTACGCGTTTTTTGTTTTTTAAGGATGAATTTGGCGGTAAAAGAGTTGGGGTTTTTAAC GGTTGTTAAGATGTAGGGGTC 110SLIT3_23619 6586 SLIT3 NM_003062 AGGGGTATTTATAGGCGTTTAGCGTTGCGGGGGATGTTTCGAGGAATCGCGCGGAGGTTTAGTTCGTGGTA GTTTACGTTGGTAGCGGAGTAGGTA 111SMPD1_24061 6609 SMPD1 NM_000543 GAAGGGTAATCGGGTGTTTTCGGCGTCGTTCGGGGTTTTGAGGGTTGGTTAGGGTTTAGGTCGGGGGGGA CGGGATAGACGAATTAG 112 SOCS1_235958651 SOCS1 NM_003745 GATAGGGTTTTGTTTTCGGCGGGTGTGGAGATAGTTGGGGCGGAGGAGGGTGTGTTAGGGCGCGTTTTAAG AGGGTTTGGCGGTAGAAAGTGGAATTCGAGGTAGCGGGGTAAAAT 113 SOX1_27153 6656 SOX1 NM_005986TTGTAGTTTTCGAGTTGGAGGTCGTTGAGGATCGAG CGTAGGAGGAAGGAGATAGCGCGTAGCGGCGGTCGGCGAGGAGATAGTATATTTCGGGTCGGGTTTAGC GTATCGTTTT 114 SOX1_27159 6656 SOX1NM_005986 GTTAGGAGTTCGTCGGTTAGCGAGTATTTGTTTTTTTTGAGTAGCGTTTTGGTTTTGCGGCGCGGTCGGTATT TGTAATTCGGGTG 115 SOX17_66072 64321SOX17 NM_22454 GAGATGTTTCGAGGGTTGCGCGGGTTTTTCGGTTCGAAGTCGTCGTTCGTGTTTTGGTTTGTCGCGGTTTGGTTTATAGCGTATTTAGGGTTTTTAGTCGGTTTAGTGA TATTGCGG 116 SPARC_Wis 6678 SPARCNM_003118 TTTCGCGGTTTTTTAGATTGTTCGGAGAGCGCGTTTTGTTTGTCGTTTGTTTGTTTGTTATTGAGGTATG 117 SPN_24052 6693 SPN NM_003123,ATCGTAGGTTGGGTTTGGTCGTTGGTAGGGAAGTG NM_001030288GGTAGAGGGGAGGTTCGGTTAGGTTTTTCGGTAATT TTCGCGTGTTTTGTTTTT 118 SST_238086750 SST NM_001048 TGGTTGCGTTGTTTATCGTTTTGGTTTTGGGTTGTGTTATCGGCGTTTTTTCGGATTTTAGATTTCGTTAGTTTTTGTAGAAGTTTTTGGTTGTTGTCGCGGGGAAGTAG GTAA 119 TAC1_56187 6863 TAC1NM_003182 GGGTATTTATTGCGACGGATAGTTTCGCGGGGTGTTGAGTTTTTTTGGTTTTTTCGAGCGTACGTTGGTCGTT TCGTATTTTCGGTAGTTGTCGTCGG 120TERT_23702 7015 TERT NM_003219, GGTTTCGATAGCGTAGTTGTTTCGGGCGGATTCGGNM_198255 GGGTTTGGGTCGCGTTTTTTCGTTCGCGCGTCGTTC GCGTTTTTAGGGTGTAG 121TFPI-2 7980 TFPI2 NM_006528 GTTCGTTGGGTAAGGCGTTCGAGAAAGCGTTTGGCGGGAGGAGGTGCGCGGTTTTTTGTTTTAGGCGGTT CGGGTGTTCGTTTTATG 122 TLL1_240517092 TLL1 NM_012464 TAAGGAATTTTGTATTCGGAGGCGGGGAGGGCGTAGGTAAATTCGGTTTTGGCGGCGTTGGCGTTCGTAGT TTGTTAGGT 123 TNFAIP1_23212 7126TNFAIP1 NM_021137 GTGGTTAGCGGATTTCGAGTCGTTTTTAGTTTGTAGTCGTTTGTTTTTTAGTAGTTTTAAGTTGTGAGTTTATATTTTGCGTTCGTCGATTTCGTTCGGAGTGTTGTTTAGTT 124 TRMT1_19794 55621 TRMT1NM_017722 TTTCGTAGGGTTCGGTGTCGTTTTTTATCGTTGTTGTATTCGGTAGTTTTGGAGATTGTTATTCGAAAAATCGG GTTTTAGAGAGTATTCGG 125 TWIST1_37291 TWIST1 NM_000474 GTTAGGGTTCGGGGGCGTTGTTCGTACGTTTCGGCGGGGAAGGAAATCGTTTCGCGTTCGTCGGAGGAAG GCGACGG 126 TWIST1_9329 7291 TWIST1NM_000474 TTTAGTTCGTTAGTTTCGTCGGTCGACGATAGTTTGAGTAATAGCGAGGAAGAGTTAGATCGGTAGTAGTCGT CGAGCGGTAAGCGCGGGGGACGTAAGCGGCGTAGTAGTA 127 UGT1A1_22912 54658 UGT1A1 NM_007120TTTTGTGGTTAGTCGCGGTAGGGGAATTTGGAGTTTTTTGGTTATTTTAGTAGAAGTTATCGATAGTTGATTG TTTATTTTACGT 128 WIF1_9096 11197WIF1 NM_007191 GCGTCGTTAGATATTTTGTTGCGTTGTAGTTTTTTTAGTTAGGGTTGTTTTCGTTTAGACGGTTGGGCGCGTC GTTTTTCGGTTTGGGTGTTA 129 WIT1_2456751352 WIT1 NM_015855 GTATGGAGCGTTTTGCGATTGTAGGAGTACGTTAGTTTTTTAGCGTTGGTTTAGTGTCGTTTGGGTTTTCGGG TATGTGGATTCGTT 130 WT1_1 7490 WT1NM_024426, TGTGTTATATCGGTTAGTTGAGAGCGCGTGTTGGGT NM_024424TGAAGAGGAGGGTGTTTTCGAGAGGGACGTTTTTTCGGATTCGTTTTTATTTTAGTTGCGAGGGCGTTTTTAA GGAGTAGCG 131 XRCC3_9322 7517XRCC3 NM_005432 CGTTTGTTTTTATAGGTTCGGGTAATGGAGATTCGCGGTCGTTTTCGTTTTTTGATTTTGTTTTATTTTTTACG TTCGTTGTCGTTTACGATTTTCGATTTCGTTGT132 ZGPAT_23961 84619 ZGPAT NM_032527TGTATGCGGAGAGGTCGTAGTTATTGTTGTGAGTAG NM_181484GATATAGTGGCGGTTGATTTGGGAGAAGTTATAGAG NM_181485GGACGGGGTGGGAGAGGGACGAGGAGTCGGGAAT GGT

TABLE 3 qMSP Molecular Beacon sequences Molecular beacon sequence(5′-3′) Row Gene Official Gene (modification beacons: 5′ FAM, 3′ DABCYL)Nr Assay Name ID Symbol Refseq SEQ ID NO's 397-425 1 ALX3_25180 257 ALX3NM_006492 CGACATGCGCGGTTGATTCGTTTTTCGGTTTGCG GGCATGTCG 2 C13orf18_Gron80183 C13orf18 NM_025113 CGACATGCCGTCGTAGGTATCGAGACGTCGTTTAGATGGGCATGTCG 3 GATA4 2626 GATA4 NM_002052CGACATGCGTAGTCGGGGTCGCGTATTTTCGTTT CGGCATGTCG 4 HOXA11_23844 3207 HOXA11NM_005523 CGACATGCGATAAAAACTCAACTCTCGTCCCCAC CGCATGTCG 5 JAM3 83700 JAM3NM_032801 CGACACGATATGGCGTTGAGGCGGTTATCGTGTCG 6 JPH3_12611 57338 JPH3NM_020655 CGTCTGCAACCGCCGACGACCGCGACGCAGACG 7 LMX1A_9513 4009 LMX1ANM_177398, CGACATGCCCGATCGCCCCCCAATACCGCATGTCG NM_177399, NM_001033507 8NOL4_19645 8715 NOL4 NM_003787 CGACATGCGGCGTTGGGCGGGCGGTTGCATGTCG 9PAK3_1 5063 PAK3 NM_002578 ACATGCCGTTTTTAGAGGGTCGGGGTTTTTTCGG CATGT 10TERT_23702 7015 TERT NM_003219, CGACATGCGACCCAAACCCCCGAATCCGCGCATGNM_198255 TCG 11 TFPI2 7980 TFPI2 NM_006528CGACATGCACCGCGCACCTCCTCCCGCCAAGCAT GTCG 12 TWIST1_3 7291 TWIST1NM_000474 CGACATGCCGGCGGGGAAGGAAATCGTTTCGCAT GTCG 13 CCNA1_Gron 8900CCNA1 NM_003914 CGACATGCACGACGCCCCCGAACCTAACGCATGT CG 14 CDO1_55929 1036CDO1 NM_001801 CGACATGCCCGACTTCCCCGAACTCCGCATGTCG 15 CDO1_55928 1036CDO1 NM_001801 CGACATGCGCGATTTCGGATTTATTGCGTTGTTAG GGCATGTCG 16GREM1_29777 26585 GREM1 NM_013372 CGACATGCGGGATTAACGTAGGCGATGTCGGGCATGTCG 17 GPNMB_52607 10457 GPNMB NM_001005340CGACATGCGGTTTTTTGGGTCGGGGCGCGGCAT GTCG 18 HIN1_3 92304 SCGB3A1 NM_052863CGACATGCAGGGTTTTTTTAGGAGCGCGGGCGAG G-GCATGTCG 19 HOXD1(2) 3231 HOXD1NM_024501 CGACATGCGGGTCGGGTTCGTCGAAGGTCGGCA TGTCG 20 LAMA1_63431 284217LAMA1 NM_005559 CGACATGCCAAAAACACGCCCCCGCGCATGTCG 21 LTB4R_31250 1241LTB4R NM_181657 CGACATGCGTAGTTTTCGTTCGCGTCGTTTGGTC GGCATGTCG 22 MAL 4118MAL NM_002371 CGACATGCAAACGAACGCCGCTCAAACTCCGCGC GCATGTCG 23 NDRG2_5660357447 NDRG2 NM_201540 CGACATGCGTTCGGTTTAGAATAGGAGATTAGTTT NM_201539AGGTTCGTTGCATGTCG NM_201535 NM_201537 24 NID2_9091 22795 NID2 NM_007361CGACATGGGTTCGTAAGGTTTGGGGTAGCGGCCA TGTCG 25 NPTX2_57779 4885 NPTX2NM_002523 CGACATGCGCGGGTAGTCGGCGTGTATCGCATGT CG 26 RASSF1A 11186 RASSF1NM_007182 CGTCTGCGTGGTTTCGTTCGGTTCGCGTTTGTTA NM_170712 GGCAGACGNM_170714 27 SALL4_12833 57167 SALL4 NM_020436CGACATGCGGAGGATTAGGGCGAGTAGTAGTCGT AGCATGTCG 28 SOX17_66072 64321 SOX17NM_22454 CGACATGCGTTCGTGTTTTGGTTTGTCGCGGTTTG GCATGTCG 29 TAC1_56187 6863TAC1 NM_003182 CGACATGCGGTTTTTTCGAGCGTACGTTGGTCGC ATGTCG

EXAMPLES Example 1 Discovery of Methylation Markers in Cervical Cancer,Using Relaxation Ranking

To identify genes that are downregulated in cervical cancer due topromoter hypermethylation and to enrich for those genes that are mostfrequently involved in cervical cancer, a multistep approach was usedcombining

-   -   Affymetrix expression microarray analysis on a panel of frozen        tissue samples from 39 human primary cervical cancers to        identify cancer-specific down-regulated genes.    -   Affymetrix expression microarray analysis on a panel of 4        different cervical cancer cell lines in which the expression of        (hyper)methylated genes was re-activated upon treatment with        5-aza-2′deoxycytidine (DAC) (blocking DNA methylation), and/or        trichostatin A (TSA) (inhibiting histone deacetylase—HDAC).

Data from both approaches were combined, and a novel non-parametricalranking and selection method was applied to identify and rank candidategenes. Using in silico promoter analysis we restricted the analysis tothose candidate genes that carry CpG-islands. The new approach resultedin a significant enrichment of hypermethylated genes: we compared thefirst 3000 high-ranking candidate probes with lists of imprinted genes,X-chromosome located genes and known methylation markers. In addition,we determined the hypermethylation status of the 10 highest rankingcandidate genes in both cervical cancers and normal cervices using COBRA(COmbined Bisulfite Restriction Analysis).

Material and Methods

Primary Cervical Tissue Samples:

For the expression microarray analysis, tissues from 39 early stagefrozen cervical cancer samples were used from a collection of primarytumors surgically removed between 1993 and 2003 (University MedicalCenter Groningen, Groningen, The Netherlands). All cervical cancerpatients underwent gynecological examination for staging in accordancewith the International Federation of Gynecology and Obstetrics (FIGO)criteria (Finan et al., 1996). Tumor samples were collected aftersurgery and stored at −80° C. The stage of cervical cancer patientsincluded 33 FIGO stage 1B (85%) and 6 FIGO stage IIA (15%). The medianage of the cervical cancer patients was 46 years (IQ range 35-52 yr.).

For COBRA and BSP (Bisulfite Sequencing PCR), 10 (of the 39) primarycervical cancers and 5 controls (normal cervix) were used. Theage-matched normal cervical controls were women without a history ofabnormal PAP smears or any form of cancer and planned to undergo ahysterectomy for benign reasons during the same period. Normal cerviceswere collected after surgery and histologically confirmed.

Informed consent was obtained from all patients participating in thisstudy.

Cervical Cancer Cell Lines:

Four cervical carcinoma cell lines were used: HeLa (cervicaladenocarcinoma, HPV18), SiHa (cervical squamous cell carcinoma, HPV16),CSCC-7 (non-keratinizing large cell cervical squamous cell carcinoma,HPV16) and CC-8 (cervical adenosquamous carcinoma, HPV45). HeLa and SiHawere obtained from the American Tissue Type Collection. CSCC-7 and CC-8(Koopman et al., 1999) were a kind gift of Prof. GJ Fleuren (LeidenUniversity Medical Center, Leiden, The Netherlands). All cell lines werecultured in DMEM/Ham's F12 supplemented with 10% fetal calf serum.

Cell lines were treated for 3 days with low to high dose (200 nM, 1 μMor 5 μM) 5-aza-2′deoxycytidine (DAC), 200 nM DAC with 300 nMtrichostatin A (TSA) after 48 hours, or left untreated. Cells were splitto low density 24 hours before treatment. Every 24 hours DAC wasrefreshed. After 72 hours cells were collected for RNA isolation.

RNA and DNA Isolation:

From the frozen biopsies, four 10-μm-thick sections were cut and usedfor standard RNA and DNA isolation. After cutting, a 3-μm-thick sectionwas stained with haematoxylin/eosin for histological examination andonly tissues with >80% tumor cells were included. Macrodissection wasperformed to enrich for epithelial cells in all normal cervices.

For DNA isolation, cells and tissue sections were dissolved in lysisbuffer and incubated overnight at 55° C. DNA was extracted usingstandard salt-chloroform extraction and ethanol precipitation for highmolecular DNA and dissolved in 250 μl TE-4 buffer (10 mM Tris; 1 mM EDTA(pH 8.0)). For quality control, genomic DNA was amplified in a multiplexPCR containing a control gene primer set resulting in products of 100,200, 300, 400 and 600 bp according to the BIOMED-2 protocol (van Dongenet al., 2003).

RNA was isolated with TRIzol reagent (Invitrogen, Breda, TheNetherlands) according to manufacturer's protocol. RNA was treated withDNAse and purified using the RNeasy mini-kit (Qiagen, Westburg, Leusden,The Netherlands). The quality and quantity of the RNA was determined byAgilent Lab-on-Chip analysis (ServiceXS, Leiden, The Netherlands,www.serviceXS.com).

Expression Data:

Gene expression for 39 primary cancers and 20 cell line samples wasperformed using the Affymetrix HGU 133 Plus 2.0 array with 54,675 probesfor analysis of over 47,000 human transcripts. The labeling of the RNA,the quality control, the microarray hybridization and scanning wereperformed by ServiceXS according to Affymetrix standards. For labeling,ten microgram of total RNA was amplified by in vitro transcription usingT7 RNA polymerase.

Quality of the microarray data was checked using histograms, boxplotsand a RNA degradation plot. One cell line sample was omitted because ofpoor quality. Using BioConductor (Gentleman et al., 2004), present (P),absent (A) or marginal (M) calls were determined with the MASSalgorithm. MASS uses a non-parametric statistical test (Wilcoxon signedrank test) that assesses whether significantly more perfect matches showmore hybridization signal than their corresponding mismatches to producethe detection call for each probe set (Liu et al., 2002). The relaxationranking approach only relied on P-calls. Some samples were analyzed induplicate, and the profile of P-calls is highly similar (93-95% of theprobesets have an identical P/M/A call).

Relaxation Ranking Algorithm:

In order to identify the most promising markers that are methylated incervical cancer, we assumed that such markers should be silenced incancer cells and upregulated upon re-activation after DAC/TSA treatment;therefore, the best methylation markers will be genes represented byprobes with:

-   -   no expression in primary cervical cancers: P-calls=0 out of 39        cancers    -   no expression in (untreated) cervical cancer cell lines:        P-calls=0 out of 4 cell lines    -   expression in cervical cancer cell lines treated with DAC (or        DAC in combination with TSA): P-calls=15 out of 15 treated cell        lines

To select for those gene probes that would be the best candidatehypermethylated genes in cervical cancer, we present the relaxationranking algorithm. Probe sets were ranked, not primarily based on thenumber of P-calls and thus explicitly setting thresholds, but primarilydriven by the number of probe sets that would be picked up, based onselection criteria (the number of P-calls in primary cancers, untreatedand treated cell lines). The stricter (e.g. P-calls: 0-0-15) theseselection criteria, the lower the number of probes that meet with thesecriteria; while if the conditions become more and more relaxed (highernumber of P-calls in primary cancers and untreated cell lines, and lowernumber of P-calls in treated cell lines), the more probes will comply.In the end, using P-calls: 39-4-0 as criteria, all probe sets werereturned. This way, there was no need to define a ‘prior’ threshold forthe number of P-calls.

The following sorting method was applied:

-   (1) All possible conditions were generated and the number of probes    that were picked up under these conditions was calculated:    -   a. the number of samples with expression (P) of a certain probe        in        -   i. primary cervical cancer samples is called x_(sample)        -   ii. cervical cancer cell lines is called y_(sample)        -   iii. treated cervical cancer cell lines is called z_(sample)    -   b. all combinations of x, y and z are made        -   i. x (the number of P-calls in primary cancers) varies from            0 to 39        -   ii. y (the number of P-calls in untreated cell lines) from 0            to 4        -   iii. z (the number of P-calls in treated cell lines) from 0            to 15        -   iv. In total, 3200 combinations of x, y and z can be made    -   c. a probeset was found under each of these generated conditions        x, y and z if:        -   i. x_(sample)≦x (number of P-calls for probe in primary            cancers smaller or equal compared to condition) AND        -   ii. y_(sample)≦y (number of P-calls for probe in untreated            cell lines smaller or equal compared to condition) AND        -   iii. z_(sample)≧z (number of P-calls for probe in treated            cell lines larger or equal compared to condition)    -   d. under very strict conditions (x=0, y=0, z=15) no probes were        found, while under the most relaxed conditions (x=39, y=4, z=0)        all probes were returned. For all combinations of x, y and z,        the number of probes that complied (w), was stored-   (2) The data was sorted with w as primary criterion (ascending),    followed by x (ascending), y (ascending) and z (descending)-   (3) This sorted dataset was analyzed row per row. In row i, the w,    probes retrieved with criteria x_(i) y_(i) z_(i) were compared with    the list of probes, already picked up in rows 1 to i−1. If a probe    did not occur in this list, it was added to the list-   (4) This process continued until there were m (user-defined) probes    in the list    DNA Methylation Analysis Using COBRA and Bisulphate Sequencing:

To validate the (hyper)methylated status of candidate gene probes, DNAextracted from 10 cervical cancers and 5 normal cervices were analyzedusing BSP and COBRA. Bisulfite modification of genomic DNA was performedusing the EZ DNA methylation kit (Zymogen, BaseClear, Leiden, TheNetherlands). The 5′ promoter region of the tested gene was amplifiedusing bisulfate treated DNA. PCR primers for amplification of specifictargets sequences are listed in Table 4. COBRA was performed directly onthe BSP products as described by Xiong et al. (Xiong and Laird, 1997)using digestions with BstUI, Taql and/or HinfI according themanufacture's protocol (New England Biolabs Inc., Beverly, Mass.). Forsequence analysis, the BSP products were purified (Qiagen) and subjectedto direct sequencing (BaseClear, Leiden, The Netherlands). Leukocyte DNAcollected from anonymous healthy volunteers and in vitro CpG methylatedDNA with SssI (CpG) methyltransferase (New England Biolabs Inc.) wereused as negative and positive control, respectively.

TABLE 4 list of primers used for BSP (¹: +1 is transcription start site(TSS); ^(2:) Several primer pairs were tested, however, none worked).Start .End .Name .Forward primer (5′-3′) .Reverse primer (5′-3′) .Taposition¹ position .RefSeq DAZL .TTTGGGGGTGATGTGTGTGTTT.TCTCCCTCAACTCACCATAATA .54 .−161 .312 NM_001351 ADARB1² .NM_015834SYCP3 AAAATTTAAAAATTGGAAGGTATT ACCTCACTAATCAAAAACAACCTCT .54 .−208 .+186NM_153694 AGG AUTS2 .TTTTAAAAGTGATAAAGTTGGTTA .CCCTTTTCTTTCTCCTCTCTTTCT56 .+300 .−184 NM_015570 TGG T NNAT .GGTTAGGGATTGGGGAGAA.GCTAAACTTACCTACAACAACAC .54 .−271 .210 NM_005386 SST.GGGGTATGTGGAATTGTGTG .AAA TCT CCT TAC CTA CTT CCC C .54 .−185 .+276NM_001048 HTRA3 .GTYGGTTTTGTYGTTATGTAGGY .AAC TTC ACT TCC TCC CTA ACC.57 .+190 .+622 NM_053044 ZFP42 AGTAGGTGTTTGTTGAAGATAG ACT CAT AAC ACACAT AAC CAT C .60 .+308 .+580 NM_174900 NPTX1 .GGTAGTGGGGGTTTGATAG.AAATAATCTCCTTCTACTACAACAC .54 .−2 .+372 NM_002522 GDA.TATAGAAGGTGGAGGAAGTTGA .CACCTCCATAAAACAAATCCAAA .54 .−239 .+194NM_004293 CCNA1 .TATAGTTGGAGTTGGAGGGT .AAACAACTAACAAATACACTAAAA .54.−279 .+146 NM_153694Results

To identify novel markers that are methylated in cervical cancer, weapplied a multistep approach that combines re-expression of silencedhypermethylated genes in cervical cancer cell lines (using DAC andDAC/TSA), downregulated expression in 39 cervical cancers expression,and selection of candidate markers using a relaxing ranking algorithm.The best profile of a candidate marker would be: no expression in any ofthe 39 cervical primary cancers and 4 untreated cancer cell lines, butre-activation of expression after demethylation and/or blocking ofhistone deacetylation in all 15 cell lines treated with variouscombinations of DAC/TSA (P-calls: 0-0-15). However, none of the probesets showed this ideal profile. To generate a list of candidate genes, arelaxation ranking algorithm was applied.

The only variable used in the relaxation ranking is the number of probeswe would like to retrieve. As shown in FIG. 1, the number of probesretrieved (w) with parameters x, y and z (the number of P-calls inrespectively primary tumor samples, untreated and treated cell lines)follows a complex profile which consists not only of additive elements,but also interactions between the parameters. In general, the number ofP-calls in primary cancer samples (x) has the largest influence on w.The sorting methodology has the advantage that no cut-off values have tobe chosen for x, y and z, and therefore there is no need to implicitlylink a relative weight factor to the parameters.

To calculate the most optimal number of potentially hypermethylatedcandidate markers for further analysis, we estimated this number basedon known (i.e. described in literature) methylation markers in cervicalcancer. Forty-five known methylation markers were found usingtext-mining using GeneCards (Rebhan et al., 1997) for aliases/symbols toquery PubMed through NCBI E-Utils. The position of the markers afterranking (“observed”) was determined as shown in the step plot in FIG. 2.If the markers would be randomly distributed in the ranking, the profilewould be similar to the curve, marked ‘expected’. This ‘expected’ curveis not a straight line, but is calculated based on whether a probe couldbe assigned with a gene symbol and taking probes into account that areassociated with a gene that is already associated with an earlierselected probe. The number of observed methylation markers has ingeneral the same slope as expected. However, until about 3000 probes,the slope of the number observed markers versus the number of selectedprobes (in dashed lines) cannot be explained if the markers would berandomly distributed as its steepness is much higher. When selectingmore than 3000 probes, the slope suddenly decreases to a level that isclose to random distribution. This enrichment can also statistically beproven. Therefore, we selected the first 3000 probes, referred to asTOP3000, in the ranking for further analysis. In this TOP3000 list, 2135probes are associated with a gene symbol, of which 1904 are unique.

Validation of the 10 Highest-Ranking Candidate Genes Using COBRA:

In order to validate whether the highest ranking genes represent markersthat are functionally hypermethylated in cervical cancer, we performedCOBRA on bisulfite-treated DNA of 10 cervical cancers and 5 normalcervices. For this analysis we focused on those first 10 genes from thehighest ranking probe-list (Table 5) that:

-   -   represent a known gene (i.e. gene symbol)    -   contain a CpG-island surrounding the TSS    -   are located on any chromosome except chromosome X    -   are expressed in less than 15 carcinomas

BSP was used to amplify the CpG-islands of these candidate genes usingbisulfite-treated DNA and COBRA to determine the methylation status.CCNA1 (at position 49) was included as a positive control for thehighest listed, reported cervical cancer specific methylation genepromoter. BSP/COBRA of CCNA1 revealed that 6 of 10 carcinomas aremethylated at the restriction enzyme sites (T1, T3, T5, T7, T9 and T10in FIG. 3). Sequence analysis of the BSP-products (on average 7-9independent clones for each carcinoma) of these 10 carcinomas revealedthat in 6 carcinomas the promoter is hypermethylated in good agreementwith the COBRA results (FIG. 3C).

TABLE 5 Methylation status using COBRA of the 10 highest ranking genepromoters. Gene selected for further validation after applyingadditional criteria. Included is CCNA1 on position 47 (original position241) as the highest ranking cervical- cancer-associated hypermethylatedgene. Methylation status was determined by BSP/COBRA (see FIG. 3 andFIG. 4). Gene Chromosomal Methylation Methylation Rank symbol locationin cancer in normal 1 DAZL 3p24.3 9/9 5/5 2 ADARB1 21q22.3 Nd Nd 3 SYCP312q 9/9 5/5 4 AUTS2 7q11.22 0/9 0/5 5 NNAT 20q11.2 9/9 5/5 6 SST 3q287/9 0/5 7 HTRA3 4p16.1 1/9 0/5 8 ZFP42 4q35.2 9/9 5/5 9 NPTX1 17q25.1 5/10 0/5 10 GDA 9q21.13 0/9 0/5 47 CCNA1  6/10 0/5

Table 5 summarizes the methylation status of the 10 highest rankinggenes in 10 cervical cancer and 5 normal cervices using COBRA. One gene(ADARB1 at rank 2) could not be analyzed for methylation as no specificBSP products could be amplified using several combinations of primerpairs. Interestingly, using the BSP products of the other 9 listedgenes, 7 (78%) showed methylation in carcinomas (Table 5). Four genesare hypermethylated in all 9 tested cancers, while for SST (7 of 9carcinomas), HTRA3 (1 of 9 carcinomas) and NPTX1 (5 of 10 carcinomas)not all tested carcinomas are hypermethylated. FIG. 4 showsrepresentative methylation analysis of 3 genes using COBRA. Three (NNAT,SST and NPTX1) of the 7 hypermethylated gene promoters have beenreported to be methylated in tumors previously. Taken these datatogether, these findings showed that the relaxation ranking algorithmresulted in a very significant enrichment for genes with a positivemethylation status.

A cervical-cancer-specific hypermethylated marker is only of relevancefor the diagnosis of (pre-) malignant disease in case normal cervicalepithelium is not methylated. COBRA analysis of 5 normal cervices forall 9 genes revealed that 4 genes (DAZL, SYCP3, ZFP42 and NNAT) arehypermethylated in all 5 samples (Table 5). On the other hand, of the 7genes hypermethylated in cervical cancer specimens, 3 genes (SST, HTRA3and NPTX1) did not show DNA methylation in any of the normal cervices of5 independent individuals. We observed the same methylation profile forCCNA1 that was reported previously as a cervical cancer specific gene(Kitkumthorn et al., 2006) with hypermethylation in only 6 of 10 tumorsbut none of the 5 normals (Table 5; FIG. 3).

Example 2 BROAD Analysis: Genome-Wide Promoter Alignment

The “Database of Transcription Start Sites” (DBTSS) (Suzuki et al.,2004) mapped each transcript sequence on the human draft genome sequenceto identify its transcriptional start site, providing more detailedinformation on distribution patterns of transcriptional start sites andadjacent regulatory regions. The promoters of the above identifiedTOP3000 genes were separately mapped on the genome-wide alignment of allpromoter associated CpG islands. All the promoter sequences weresubsequently aligned by clustalW algorithm (Li 2003; Thompson et al.,1994). Treeillustrator (Trooskens et al., 2005) was used to visualizethe large guide tree in addition to indicating the location of the knownmarkers. Some regions on the “circle” are denser in known markers thanothers, indicating that there might be a sequence mechanism located inthe small region around the TSS which makes certain genes moremethylation-prone. The genes were selected as candidates to bemethylated if they were located in a cluster, i.e. less than 9 nodes(distance to the closest neighboring marker) away from a marker alreadydescribed in the literature. These genes were assigned a score,calculated as follows: if the gene is a known literature marker, score+10, if a known marker is one node away, score +9, if there are markerstwo nodes away: addition to score=number of markers*8, etc. The geneswere ranked according to this score.

A final gene selection was made based on the ranking, the opportunity todesign primers, genes to be known as tumor suppressor genes and expertknowledge on their function, history and mutation status in other cancertypes. Also known genes from literature and previous research wereincluded for confirmation.

A final selection of markers resulting from the above set outapproaches, were tested on tissue using the Base5 methylation profilingplatform (Straub et al. 2007). Differential methylation of theparticular genes was assessed using Base5 methylation profiling platformas follows: DNA was extracted from cervical samples, bisulfiteconverted, and selected regions of the particular genes were amplifiedusing primers whose sequence represented converted or non-converted DNAsequences. Amplification was monitored in real-time set up usingSYBRgreen. Data analyses designed to cope with inherent variance (i.e.,noise) in measured Ct and Tm values were applied to withhold 112different assays for detecting differential methylation of ALX3, ALX4,AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf48,C9orf19, CALCA, CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B,CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2,EGFR, EGR4, EPB41L3, FOS, FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, Gst-Pi,HHIP, HOOK2, HOXA1, HOXA11, HOXA7, IGSF4, ISYNA1, JAM3, JPH3, KNDC1,KRAS, LMX1A, LOC285016, LOX, MTAP, MYO18B, NOL4, NPTX1, OGFOD2, PAK3,PAX1, PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, RARA, RBP4, RECK, RPRM,SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, SOCS1, SOX1, SPARC, SPN,SST, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1,WT1, XRCC3, and ZGPAT in cervical cancer tissue samples.

Material and Methods

Samples:

A total of 201 frozen tissue samples (87 cervical cancer samples, themajority derived from squamous cell carcinomas; and 114 normal tissues)were collected by UMC Groningen. If the tissue contained more than 20%stromal cells, the samples were macro-dissected to enrich for tumorcells.

DNA Isolation and Bisulphite Modification:

DNA was isolated using proteinase K digestion and phenol/chloroformextraction. DNA concentration was measured using NanoDropSpectrophotometer. From each sample, up to 2 μg of genomic DNA wasconverted using a bisulphite based protocol (EZ DNA Methylation Kit™,ZYMO Research, Orange, Calif.).

Detection of Hypermethylation:

Methylation specific PCR (MSP) primers were designed for each of thegenes assessed for (hyper)methylation. An example on primer designspanning a large region of the promoter is provided in FIGS. 5A and 5Bfor ALX4.

For some genes more primer pairs were designed giving a total of 424different assays. These assays were applied on 8 sub-arrays of 2OpenArray™ plates by BioTrove Inc. The beta-actin assay was applied oneach sub-array as an internal control. Quality control was performedusing an in vitro methylated DNA sample and a negative control sample.The selectivity and the reproducibility were checked. After DNAconversion and purification, beta-actin copy number was determined byqMSP. The equivalent of 1500 beta-actin copies per sample was appliedper sub-array of an OpenArray™ plate on a real-time qPCR system(BioTrove Inc.) using the DNA double strand-specific dye SYBRgreen forsignal detection.

The cycling conditions were: 90° C.-10 seconds, (43° C. 18 seconds, 49°C. 60 seconds, 77° C. 22 seconds, 72° C. 70 seconds, 95° C. 28 seconds)for 40 cycles, 70° C. for 200 seconds, 45° C. for 5 seconds. A meltingcurve was generated in a temperature range between 45° C. and 94° C.

Analysis of Methylation:

For each combination of assays and samples two parameters were collectedusing an algorithm which is part of the standard data analysis packageoffered by the supplier. The parameters were the Ct value (thresholdcycle number) of the assessed amplicon and the melting temperature ofthe assessed amplicon. The following data analysis workflow was appliedto the results created by the software which came with the systemOpenArray™ system: Data was collected for each combination of assays andsamples in the two sets of samples used. Results were filtered using thefollowing approach. Read outs from not loaded reaction spaces wereremoved from analysis. Technical Control assays were removed from thedata set. Assays known to not work for other than biological reasonswere removed from the analysis. Per sub-array, signals were onlyinterpreted if there was a positive beta-Actin call. Ct values >0 foreach gene were normalized using the Ct values collected for the genebeta-Actin. This resulted in two files containing the results for eachset of sample. 201 samples were tested of which 6 gave invalid results.In total 79,170 reactions were performed of which 74,110 were valid. Forthe data analysis, 2 boundaries were defined: an upper bound onbeta-Actin-normalized-Ct (banCt) and a lower bound on MeltingTemperature (Tm). Samples below the banCt boundary and above the Tmboundary are considered to be “methylated”, others (including allsamples with no signal, i.e. Ct>40) are classified as “unmethylated”. Inboth dimensions the set of candidate boundaries consists of all valuesin between 2 measurements, plus infinity (the equivalent of noboundary). The set of candidate models for “methylated” then consists ofall combinations of candidate Tm lower bound and a banCt upperbound. Ascore is computed for each of these candidate models, as follows. Count:cancers inside boundaries=true positives (TP), cancers outsideboundaries=false negatives (FN), normals inside boundaries=falsepositives (FP), normals outside boundaries=true negatives (TN). Abinomial test was applied to find out how unusual it is to have at leastTP successes in (TP+FP) trials where the probability of success is(TP+FN). The lower this probability value is the better. Then qualitycontrol data were taken into account to determine the most robustboundaries. Using the standard deviations (StDevQC) observed in the QC,a series of increasingly “noisy” datasets were generated. Themeasurements are replaced by a value randomly selected from a normaldistribution with average equal to the observed measurement and standarddeviation equal to StDevQC multiplied by a value that gradually (10noise levels) increases from 0 to 2. Each time the score of thecandidate model is computed by applying the 2 steps above (i.e., countand binomial test). All these scores (11 in total: 1 for “no noise” and10 for noise levels 0.2, 0.4, . . . , 2) are added up to obtain theultimate accumulated score. The candidate model with the best (i.e.lowest) accumulated score is retained. This same score of the bestcandidate model for each marker is also used for ranking the markers.

Results

A high throughput, real-time methylation specific detection platform wasapplied on two groups of samples isolated from cervical cancer tissueand from corresponding normal cervical tissue. In this study it wasshown that a number of genes are differentially methylated in cervicalcancer. We identified 112 different assays for detecting 96 differentgenes being differentially methylated in human cervical cancer tissueand normal cervical tissue control samples. The genes identified areALX3, ALX4, AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, BMP6, BNIP3,C13orf18, C16orf48, C9orf19, CALCA, CAMK4, CCNA1, CCND2, CDH1, CDH4,CDK6, CDKN1B, CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, CYCLIND2,DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4, EPB41L3, FOS, FOXE1, GADD45A,GATA4, GDAP1L1, GNB4, Gst-Pi, HHIP, HOOK2, HOXA1, HOXA11, HOXA7, IGSF4,ISYNA1, JAM3, JPH3, KNDC1, KRAS, LMX1A, LOC285016, LOX, MTAP, MYO18B,NOL4, NPTX1, OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC, PRKCE, RAD23B,RALY, RARA, RBP4, RECK, RPRM, SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3,SMPD1, SOCS1, SOX1, SPARC, SPN, SST, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1,TWIST1, UGT1A1, WIF1, WIT1, WT1, XRCC3, and ZGPAT

The resulting assays have the assay details provided in Table 1, Table2, and FIG. 5B.

Example 3 Further Assay Selection: Base 5—Lightcycler Platform

Of the different assays listed in Table 1 previously identified usingthe Base5 methylation platform, the top 63 ranked assays plus β-actin(ACTB) were transferred to the Lightcycler platform in order to furtherfine-tune the selection of the best cervical cancer methylation markers.This platform allows the assessment of markers in a system which iscloser to, and provides information valuable for the subsequentdevelopment of, a final, scaled up MSP assay. The 64 assays (Table 6)were applied on a 384 well plate by Sigma. Six repeats of the assay setfitted on a 384 well plate. The samples were randomized per plate.

The sample set selected for the Lightcycler analysis was also previouslyused in the Base 5 analysis in order to make a compared analysis: atotal of 27 cervical tumor samples and 20 controls (frozen tissue) werecollected by UMC Groningen.

TABLE 6 The 64 selected assays which were applied on the Lightcyclerplatform N° Assays Base 5 ranking 1 LMX1A_9513 1 2 SLIT2_23681 2 3ISYNA1_19726 3 4 EPB41L3_19071 4 5 WT1_1 5 6 DKK2_23973 6 7 ALX3_25180 78 JAM3 8 9 JPH3_12611 9 10 SLIT2_23672 10 11 SOX1_27153 11 12 SOX1_2715912 13 RALY_19607 13 14 RPRM_2 14 15 CDH4_24735 15 16 CPT1C_23912 16 17SLIT2_23676 17 18 PAX1_27211 18 19 DKK2_23970 19 20 TERT_23702 20 21NOL4_19645 21 22 HOXA11_23844 22 23 CALCA_2 23 24 C13orf18_19885 24 25PAX1_27210 25 26 WIT1_24567 26 27 GATA4_13295 27 28 SLIT1_23651 28 29LOC285016_22940 29 30 POMC 30 31 Gst-Pi_New3 32 32 DAPK1 34 33GDAP1L1_19773 35 34 TFPI-2 36 35 TWIST1_9329 37 36 SST_23808 38 37EGR4_24277 39 38 C16orf48_22922 45 39 DBC1_23879 46 40 GDAP1L1_19775 4741 OGFOD2_23131 48 42 ALX4_25062 49 43 TLL1_24051 51 44 CTDSPL_23795 5245 CYCLIND2_1 58 46 COL1A1_23253 65 47 CDK6_9703 71 48 CDH1_17968 76 49SOCS1_23595 78 50 FOXE1_13314 91 51 BMP2_17901 94 52 AURKA_24802 110 53SEMA3F_23485 120 54 PAK3_3 121 55 HOXA7_2 125 56 CTDSPL_23804 127 57NPTX1_2 136 58 SLIT1_23653 164 59 SMPD1_24061 174 60 GADD45A_24463 25061 KRAS_24235 281 62 RECK_18940 321 63 UGT1A1_22912 341 64 Beta_ActinInternal control

Tissue slides were deparaffinized using 100% xylene followed by 100%ethanol. Pellet was resuspended in a buffer containing SDS-proteinase K,and DNA was extracted with phenol-chloroform followed by ethanolprecipitation. DNA concentration was measured using NanoDropSpectrophotometer. From each sample, up to 3 μg of genomic DNA wasconverted using a bisulphite based protocol (EZ DNA Methylation Kit™,ZYMO Research). After DNA conversion and purification, equivalent of 20ng of gDNA was used per reaction. All the samples were tested onLightcycler using Sybergreen as detector and the amplicon size wasdetermined by capillary electrophoresis.

Quality control was performed using in vitro methylated DNA sample,unmethylated DNA sample (Chemicon International, CA, USA; Cat.#57821 andCat.#57822) and no template control sample (H₂O). From the Lightcyclerplatform, the Ct values (cycle number at which the amplification curvescross the threshold value, set automatically by the software) andmelting curves (Tm) were generated. From the capillary electrophoresisplatform, size of the amplicon and intensity of the signal detected weregenerated. For each assay, Tm and amplicon size parameters weredetermined in in vitro methylated DNA sample, unmethylated DNA sampleand no template control sample. The measured Tm and amplicon size valueswere compared to the calculated values. If the Tm or amplicon sizevalues were out of the range of the calculated ones, the assay wasconsidered as non specific and disqualified. All the 64 assays werespecific.

A sample is considered methylated if Ct is under 40 and if Tm andamplicon size are within the boundaries of Tm+/−2 degrees and ampliconsize+/−10 bp. The intensity of the band detected by capillaryelectrophoresis had to be higher than 20. Those evaluation criteria havebeen developed based on concordance with existing Molecular Beacon basedqMSP assays, to ensure that the conclusions drawn from these data wouldbe predictive of MSP assays developed subsequently.

DNA methylation calls were compared between cervical cancer and controlpatients. An assay ranking with the set of samples was generated and theresults are summarized in the methylation table of FIG. 6. A one-tailedFisher's exact test was used as a scoring function to rank the candidatemarkers. The calculation of Fisher's exact test was based on a formulaas described by Haseeb Ahmad Khan in “A visual basic software forcomputing Fisher's exact probability” (Journal of Statistical Software,vol. 08, issue i21, 2003).

A comparison between the results coming from the Base 5 (Biotrove) andthe Lightcycler platforms has been performed. Most of the interestingassays discovered on the Base 5 platform were confirmed on theLightcycler platform.

Example 4 QMSP

Seventeen assays (ALX3, C13ORF18, DBC1, EPB41L3, GATA4, HOXA11, JAM3,JPH3, LMX1A, NOL4, PAK3, SLIT2_23672, SLIT2_23676, SOX1, TERT, TFPI2 andTWIST1_3) were further selected based on their performance on theBiotrove and Lightcycler platforms and on complementarity analysis tomaximize discriminatory power. For these assays, qMSPs using MolecularBeacon as detection system were designed (3 designs, if possible, wereevaluated per assay) and tested on control samples. For this selection,assays were judged on several criteria, including backgroundfluorescence, dynamic of the curve, and level of fluorescence generated.PCR material was used for generating standard curves for quantificationof the results.

Five assays did not meet the desired specifications (EPB41L3, SOX1,SLIT2_23672, DBC1, and SLIT2_23676) and may be redesigned in a laterphase or can be used on another detection platform. The remaining 12assays were further tested on converted DNA of cervix cancer cell lines.

All these results were taken into account to decide which assays shouldbe further verified on cervical tissue samples collected by Ulg (normalPE tissue samples #13, cancer PE tissue samples #17) and/or UMCG (normalfrozen tissue samples #20, cancer frozen tissue samples #27).

Seventeen (CCNA1, CDO1_55928, CDO1_55929, GREM1, GPNMB, HIN1, HOXD1,LAMA1, LTB4R, MAL, NDRG2, NID2, NPTX2, RASSF1A, SALL4, SOX17, and TAC1)additional good performing assays were also selected for furtherverification on the cervix tissue samples. These candidates were takenfrom other in-house cancer projects, and were not tested on theBiotrove/Lightcycler platform as described above.

DNA was isolated from the cervix tissue samples using aphenol-chloroform procedure, quantified using the picogreen method and1.5 μg of DNA was bisulphite treated using the ZYMO kit.

qMSPs were carried out in a total volume of 12 μl in 384 well plates inan ABI PRISM 7900HT instrument (Applied Biosystems). The final reactionmixture consisted of in-house qMSP buffer (including 80.4 nmol ofMgCl2), 60 nmol of each dNTP, 0.5 U of Jump Start Taq polymerase(SIGMA), 72 ng of forward primer, 216 ng of reverse primer, 1.92 pmol ofMolecular Beacon detection probe, 6.0 pmol of ROX (passive referencedye) and 72 ng of bisulphite converted genomic DNA. Thermal cycling wasinitiated with an incubation step of 5 minutes at 95° C., followed by 45cycles (95° C. for 30 seconds, 57° C. for 30 seconds, 72° C. for 30seconds). A finalizing step was performed at 72° C. for 5 minutes toconclude cycling. These conditions were similar for all the test genesas well as for ACTB. Cell lines [in vitro methylated DNA sample andunmethylated DNA sample (Chemicon International, CA, USA; Cat.#57821 andCat.# S7822)] were included in each run as positive and negativecontrols, and entered the procedure at the DNA extraction step. Primersand molecular beacon sequences used for the different qMSPs aresummarized in Table 1 and Table 3. Corresponding amplicons aresummarized in Table 2.

Ct values were determined using the SDS software (version 2.2.2.)supplied by Applied Biosystems with automatic baseline settings andthreshold. The slopes and R² values for the different standard curveswere determined after exporting data into MS Excel.

As an example, FIG. 7 shows the amplification plot obtained for thestandard curve for TAC1_56187 (960000 copies to 9.6 copies of the gene)and FIG. 8 shows the amplification plot obtained for the standard curveand for all samples for TAC1_56187. The Ct values plotted against theLog Copies of TAC1_56187 (FIG. 9) give a R² of 0.9995 and the efficiencyof the reaction is 99.35%.

In addition to the test genes, the independent reference gene β-actin(ACTB) was also measured. The ratios between the test genes and ACTBwere calculated to generate the test result. The samples were classifiedas methylated, unmethylated, or invalid based on the decision tree shownin FIG. 10.

A provisional cut-off was defined for each gene, chosen based on thegreater of either the highest value seen among the controls or a value 3times the standard deviation of the values from control samples.

The one-tailed Fisher's exact test as described above was used as ascoring function to rank the candidate markers (Journal of StatisticalSoftware, vol. 08, issue i21, 2003).

Table 7 summarizes the results obtained for TAC1_56187. Table 8summarizes the results obtained for all the tested markers on tissuesamples. The individual performances of the assays are shown in FIG. 11and the assays are ranked according their p-value (Fisher's exact test).The best performing markers were further tested on clinical samples(scrapings).

TABLE 7 Summary of the test results for TAC1_56187 on cervix tissuesamples. In column “methylation status”, the black boxes indicate themethylated results; white boxes indicate the unmethylated results.

TABLE 8 Summary of the performance results of all the tested markers ontissue samples. Ranking NA 12 NA 3 1 NA NA NA 21 8 7 10 NA NALightcycler Ranking NA 1 NA 21 8 NA NA NA 27 20 9 22 NA NA Base5 Ranking1 2 3 4 5 6 7 8 9 10 11 12 13 14 qMSP tissue NID2_(—) LMX1A_(—) TAC1_(—)NOL4_(—) CDO1_(—) CDO1_(—) SOX17_(—) TERT_(—) HOXA11_(—) LAMA1_(—)CCNA1_(—) Assays 9091 9513 56187 19645 JAM3 55929 55928 66072 GATA423702 JPH3 23844 63431 Gron Sensitivity 95 85 85 91 83 88 81 80 71 65 7265 59 60 Specificity 97 100 100 95 100 100 97 97 97 100 97 100 100 97Cut off 2 10 25 2 5 0 20 35 2 1 1 20 5 15 RatioMax 2 10 22 2 5 0 28 51 21 1 20 3 15 (Normals) STDEV 1.5 6.5 18.0 1.9 3.5 0.0 17.7 32.0 1.3 0.50.6 14.2 2.2 11.6 (Normals) *3 Cncr Meth+ 41 39 39 42 38 38 38 37 42 3933 28 27 36 Cncr Meth− 2 7 7 4 8 5 9 9 17 21 13 15 19 24 Cntrl Meth+ 1 00 2 0 0 1 1 1 0 1 0 0 1 Cntrl Meth− 31 38 38 36 38 32 37 37 38 38 37 3238 37 p-value 1.42E−17 3.94E−17 3.94E−17 7.55E−17 2.27E−16 2.79E−162.08E−14 3.66E−14 8.90E−13 2.40E−12 1.06E−11 4.82E−10 5.53E−10 1.03E−09(Fisher test) Ranking 15 NA NA 16 NA NA NA 25 NA NA 6 NA NA NALightcycler Ranking 24 NA NA 36 NA NA NA 7 NA NA 121 NA NA NA BiotroveRanking 15 16 17 18 19 20 21 22 23 24 25 26 27 28 qMSP tissueC13ORF18_(—) GREM1_(—) GPNMB_(—) NDRG2_(—) NPTX2_(—) ALX3_(—) SALL4_(—)HOXD1 LTB4R_(—) Assays Gron MAL 29777 TFPI2 52607 56603 57779 25180HIN1_3 12833 PAK3_1 (2) 31250 RASSF1a Sensitivity 65 53 51 60 50 59 5555 50 33 30 30 60 45 Specificity 97 100 97 100 100 95 100 100 100 97 10095 68 74 Cut off 2 1 2 5 5 5 30 5 1 20 10 50 20 0 RatioMax 3 0 3 5 4 629 3 0 35 6 59 28 0 (Normals) STDEV 1.8 0.3 1.8 3.1 2.7 4.5 24.3 2.0 0.218.5 4.2 45.2 17.9 0.2 (Normals) *3 Cncr Meth+ 28 23 22 12 17 20 11 1110 14 6 6 12 9 Cncr Meth− 15 20 21 8 17 14 9 9 10 29 14 14 8 11 CntrlMeth+ 1 0 1 0 0 1 0 0 0 1 0 1 6 5 Cntrl Meth− 31 32 31 19 19 18 19 19 1932 19 18 13 14 p-value 9.66E−09 8.07E−08 2.91E−06 3.22E−05 7.22E−058.61E−05 1.00E−04 1.00E−04 2.91E−04 9.64E−04 1.19E−02 5.29E−02 7.19E−021.89E−01 (Fisher test)

Example 5 Best Performing Markers Tested on Clinical Cervical ScrapingSamples

Cervical scraping samples were collected under the Cervical CancerClinical Collaborative Research Agreement study of ONCO with theGynecology Department of the UMCG hospital. The scraping samples weretaken from patients who were referred to the hospital with an abnormalPAP smear or because they were suspected for cervical carcinoma.Gynecological examination under general anesthesia was performed in allcervical cancer patients for staging in accordance with theInternational Federation of Gynecology and Obstetrics (FIGO) criteria.Control scraping samples were taken from women who visited the hospitalfor a non-malignant condition, e.g. fibroids, prolaps uteri orhypermenorrhea, and who were scheduled to undergo a hysterectomy. Whilethe patient was under general anesthesia, the cervix was scraped with anAyres spatula and brush. The scraped cells were suspended in 5-ml PBS.Cytospins for cytomorphological assessment were made (⅕ volume).Cytospins were Papanicolaou stained and routinely classified accordingto a modified Papanicolaou system (Hanselaar AG. Kwaliteit vancytopathologisch onderzoek in het herziene bevolkingsonderzoek naarbaarmoederhalskanker. Nederlands Tijdschrift voor Obstetrie enGynaecologie 1996; 109:207-210) without knowledge of the clinical data.The remaining 4-ml of the scraped cells was centrifuged, washed,aliquoted, snap-frozen in liquid nitrogen and stored at −80° C. DNA wasextracted using standard salt-chloroform extraction and ethanolprecipitation. DNA of the pellet was used for qMSP of a panel of goodperforming markers for cervical cancer and also for HPV typing.

DNA was extracted from the scraped cells using standard salt-chloroformextraction and ethanol precipitation for high molecular DNA, dissolvedin 250 μL TE-4 buffer (10 mM Tris; 1 mM EDTA, pH 8.0) and kept at −20°C. until tested.

Presence of high risk HPV was analyzed by PCR using HPV16 and HPV18specific primers on DNA of the scraping samples. On all HPV16- orHPV18-negative cases, general primer-mediated PCR was performed usingtwo HPV consensus primer sets, CPI/CPIIG and GPS+/6+, with subsequentnucleotide sequence analysis, as described previously [by Wisman et alInt j cancer 2006].

qMSP was performed after bisulphite treatment on denatured genomic DNA.The assays were carried out as described above. The samples wereclassified as methylated, unmethylated, or invalid as described above.The results obtained for all the tested markers on scraping samples fromcervical cancer patients and from control patients were ranked accordingtheir p-value (Fisher's exact test) (Table 9). Some markers have ahigher sensitivity for squamous cell carcinoma than for adenocarcinoma(NID2, JPH3, CCNA1) and some markers have a higher sensitivity foradenocarcinoma than for squamous cell carcinoma (JAMS, CDO1, HOXA11).

Various combinations of markers were evaluated to see if such acombination could increase the sensitivity while still maintaining ahigh level of specificity. In all cases, if any marker of a combinationpanel was positive, the sample was classified as methylated. Examples ofthe performance of combination of markers are summarized in Table 10. Itcan be seen that several combinations provided a sensitivity andspecificity greater than 90%.

TABLE 9 Summary of the results obtained for all the tested markers onscraping samples from cervical cancer patients and from control patients(Sens: sensitivity; SCC: squamous cell carcinoma; Ade: adenocarcinoma;cncr: cancer; ctrl: control). NID2_(—) CDO1_(—) CDO1_(—) LMX1A_(—)TAC1_(—) GREM1_(—) HOXA11_(—) JAM3 9091 55928 55929 9513 56187 2977723844 JPH3 Sensitivity 81.0% 78.5% 82.3% 78.5% 75.9% 72.2% 72.2% 62.0%64.6% Specificity 98.6% 98.6% 95.7% 97.1% 97.1% 98.6% 97.1% 100.0% 98.6%Sens SCC 80.3% 83.3% 81.8% 77.3% 77.3% 72.7% 72.7% 59.1% 69.7% Sens Ade84.6% 53.8% 84.6% 84.6% 69.2% 69.2% 69.2% 76.9% 38.5% cncr test+ 64 6265 62 60 57 57 49 51 cncr test− 15 17 14 17 19 22 22 30 28 ctrl test+ 11 3 2 2 1 2 0 1 ctrl test− 68 68 66 67 67 68 67 69 68 SCC test+ 53 55 5451 51 48 48 39 46 SCC test− 13 11 12 15 15 18 18 27 20 Ade test+ 11 7 1111 9 9 9 10 5 Ade test− 2 6 2 2 4 4 4 3 8 cncr/ctrl 4.75E−26 1.21E−244.57E−24 3.11E−23 6.24E−22 1.86E−21 4.17E−20 1.23E−18 4.12E−18 p-val5.32E−01 2.31E−02 5.84E−01 4.33E−01 3.37E−01 4.79E−01 4.79E−01 1.86E−013.53E−02 Ade/SCC Cut off 2 5 5 35 15 15 10 1 5 C13orf18_(—) CCNA1_(—)TERT_(—) NDRG2_(—) NOL4_(—) LAMA1_(—) GATA-4 Gron Gron 23702 56603 1964563431 Sensitivity 62.0% 53.2% 51.9% 58.2% 49.4% 43.0% 51.9% Specificity97.1% 100.0% 100.0% 97.1% 98.6% 98.6% 94.2% Sens SCC 62.1% 54.5% 57.6%60.6% 48.5% 43.9% 50.0% Sens Ade 61.5% 46.2% 23.1% 46.2% 53.8% 38.5%61.5% cncr test+ 49 42 41 46 39 34 41 cncr test− 30 37 38 33 40 45 38ctrl test+ 2 0 0 2 1 1 4 ctrl test− 67 69 69 67 68 68 65 SCC test+ 41 3638 40 32 29 33 SCC test− 25 30 28 26 34 37 33 Ade test+ 8 6 3 6 7 5 8Ade test− 5 7 10 7 6 8 5 cncr/ctrl 7.73E−16 2.91E−15 8.21E−15 2.03E−141.59E−12 1.61E−10 2 17E−10 p-val 6.43E−01 4.00E−01 2.33E−02 2.54E−014.80E−01 4.81E−01 3.25E−01 Ade/SCC Cut off 2 0 1 5 150 5 10

TABLE 10 Examples of the performance of combination of markers onscraping samples from cervical cancer patients and from control patients(Sens: sensitivity; SCC: squamous cell carcinoma; Ade: adenocarcinoma;cncr: cancer; ctrl: control). JAM3\ JAM3\ JAM3\ CDO1_55929\ JAM3\NID2_9091\ TAC1_56187\ NID2_9091\ HOXA11_23844\ JAM3\ HOXA11_23844\HOXA11_23844\ HOXA11_23844\ HOXA11_23844 CCNA1_Gron HOXA11_23844GREM1_29777 CDO1_55929 CDO1_55929 Sensitivity 89.9% 92.4% 88.6% 91.1%92.4% 92.4% Specificity 98.6% 95.7% 98.6% 95.7% 94.2% 94.2% Sens SCC92.4% 92.4% 89.4% 92.4% 92.4% 92.4% Sens Ade 76.9% 92.3% 84.6% 84.6%92.3% 92.3% cncr test+ 71 73 70 72 73 73 cncr test− 8 6 9 7 6 6 ctrltest+ 1 3 1 3 4 4 ctrl test− 68 66 68 66 65 65 SCC test+ 61 61 59 61 6161 SCC test− 5 5 7 5 5 5 Ade test+ 10 12 11 11 12 12 Ade test− 3 1 2 2 11 p-val 8.14E−32 6.60E−31 6.87E−31 6.62E−30 1.17E−29 1.17E−29 cncr/ctrlJAM3\ HOXA11_23844\ JAM3\ JAM3\ NID2_9091\ CDO1_55929 CDO1_55928NID2_9091 CDO1_55928 Sensitivity 92.4% 89.9% 86.1% 88.6% Specificity94.2% 94.2% 97.1% 94.2% Sens SCC 92.4% 89.4% 86.4% 89.4% Sens Ade 92.3%92.3% 84.6% 84.6% cncr test+ 73 71 68 70 cncr test− 6 8 11 9 ctrl test+4 4 2 4 ctrl test− 65 65 67 65 SCC test+ 61 59 57 59 SCC test− 5 7 9 7Ade test+ 12 12 11 11 Ade test− 1 1 2 2 p-val 1.17E−29 9.85E−28 1.13E−277.67E−27 cncr/ctrl

HPV testing will certainly continue to occupy a significant position inthe diagnosis of cervical cancer. With this in mind, the best performingmethylation markers were tested on scraping samples from patients whowere referred to the hospital with an abnormal Pap smear and thesesamples were also tested for hr HPV and HPV16. The provisional cut offas defined above was reduced in order to obtain the highest possiblesensitivity and specificity compared to the performance of hrHPV. Theresults of these tests are shown in Table 11. For these testing, theclassification of pre-cancerous (CIN) conditions were used. Sensitivitywas calculated for samples indicating cancer, CIN 2 and CIN 3, whilespecificity was calculated for those samples from controls, and thoseindicating CIN 1 or CIN 0 after cytological examination. Overall thespecificity of the methylation markers was higher compared to hr-HPV orHPV16 testing but with a lower sensitivity. Combinations of methylationmarkers (where at least one of the markers scores positive) showed acomparable sensitivity and specificity for cancers and controls, but amuch higher specificity for CIN0 and CIN1. The sensitivity for CIN3 andCIN2 is however somewhat lower. In order to increase the sensitivity forCIN3 and CIN2 detection, an analysis was made of combining the resultsof methylation markers and HPV16 (Table 12). The sensitivity as well asthe specificity increased if HPV16 was combined with the methylationmarkers.

TABLE 11 Overall summary of the methylation marker(s) results onscraping samples from patients who were referred to the hospital with anabnormal Pap smear, and from cervical cancer and control patients.(Sens: sensitivity; Spec: specificity; CIN0, CIN1, CIN2, CIN3: cervicalintraepithelial neoplasia grade 0, 1, 2, and 3; cncr: cancer; ctrl:control, NA: not applicable). hr-HPV HPV16 JAM3 NID2_9091 LMX1A_9513CDO1_55928 TAC1_56187 C13ORF18_Gron Sens Cncr  90%  77%  83%  80%  82% 83%  73%  54% Sens CIN3  95%  83%  38%  40%  60%  43%  17%  24% SensCIN2  74%  45%  21%  29%  29%  24%   7%   5% Spec Cntrl  96%  99%  99% 93%  94%  91%  93%  100% Spec CIN0  51%  91%  98%  95%  91%  98%  100% 100% Spec CIN1  34%  78%  98%  93%  85%  88%  100%  98% Overall  87% 70%  56%  57%  63%  58%  42%  34% sens Overall  67%  91%  98%  93%  91% 92%  97%  99% spec Cut off NA NA  1  2 10  3 10  0 cncr test+ 74 63 6866 67 68 60 44 cncr test−  8 19 14 16 15 14 22 38 CIN3 test+ 40 35 16 1725 18  7 10 CIN3 test−  2  7 26 25 17 24 35 32 CIN2 test+ 31 19  9 12 1210  3  2 CIN2 test− 11 23 33 30 30 32 39 40 ctrl test+  3  1  1  5  4  6 5  0 ctrl test− 66 68 68 64 65 63 64 69 CIN0 test+ 21  4  1  2  4  1  0 0 CIN0 test− 22 39 42 41 39 42 43 43 CIN1 test+ 27  9  1  3  6  5  0  1CIN1 test− 14 32 40 38 35 36 41 40 JAM3\ JAM3\ JAM3\ JAM3\ CDO1_55928\NID2\ NID2_9091 LMX1A_9513 CDO1_55928 NID2_9091 LMX1A_9513 Sens Cncr 88%  87%  90% 91%  88% Sens CIN3  45%  62%  52% 57%  67% Sens CIN2  33% 36%  31% 38%  43% Spec Cntrl  93%  93%  90% 84%  88% Spec CIN0  95% 93%  98% 95%  91% Spec CIN1  93%  83%  88% 83%  83% Overall  63%  67% 66% 69%  71% sens Overall  93%  90%  92% 87%  88% spec Cut off NA NA NANA NA cncr test+ 72 71 74 75  72 cncr test− 10 11  8 7  10 CIN3 test+ 1926 22 24  28 CIN3 test− 23 16 20 18  14 CIN2 test+ 14 15 13 16  18 CIN2test− 28 27 29 26  24 ctrl test+  5  5  7 11   8 ctrl test− 64 64 62 58 61 CIN0 test+  2  3  1 2   4 CIN0 test− 41 40 42 41  39 CIN1 test+  3  7 5 7   7 CIN1 test− 38 34 36 34  34

TABLE 12 Overall summary results of methylation marker(s) in combinationwith HPV16 on scraping samples from patients who were referred to thehospital with an abnormal Pap smear, and from cervical cancer andcontrol patients. (Sens: sensitivity; Spec: specificity; CIN0, CIN1,CIN2, CIN3: cervical intraepithelial neoplasia grade 0, 1, 2, and 3;cncr: cancer; ctrl: control). JAM3\ NID2_9091\ LMX1A_9513\ CDO1_55928\PAC1_56187\ C13ORF18_Gron\ hr-HPV HPV16 HPV16 HPV16 HPV16 HPV16 HPV16HPV16 Sens Cncr  90%  77% 95%  93%  94%  99% 93%  83% Sens CIN3  95% 83% 88%  90%  88%  88% 83%  86% Sens CIN2  74%  45% 60%  62%  60%  60%50%  45% Spec Cntrl  96%  99% 97%  93%  93%  90% 91%  99% Spec CIN0  51% 91% 88%  86%  84%  88% 91%  91% Spec CIN1  34%  78% 78%  73%  66%  73%78%  76% CIN0 test− 22 39 38  37 36 38 39  39 CIN0 test+ 21  4 5   6  7 5 4   4 CIN1 test− 14 32 32  30 27 30 32  31 CIN1 test+ 27  9 9  11 1411 9  10 CIN2 test− 11 23 17  16 17 17 21  23 CIN2 test+ 31 19 25  26 2525 21  19 CIN3 test−  2  7 5   4  5  5 7   6 CIN3 test+ 40 35 37  38 3737 35  36 cncr test−  8 19 4   6  5  1 6  14 cncr test+ 74 63 78  76 7781 76  68 ctrl test− 66 68 67  64 64 62 63  68 ctrl test+  3  1 2   5  5 7 6   1 Overall  87%  70% 84%  84%  84%  86% 80%  74% sens Overall  67% 91% 90%  86%  83%  85% 88%  90% spec JAM3\ JAM3\ JAM3\ JAM3\CDO1_55928\ NID2\ NID2_9091\ LMX1A_9513\ CDO1_55928\ NID2_9091\LMX1A_9513\ HPV16 HPV16 HPV16 HPV16 HPV16 Sens Cncr  98%  96%  100% 100%  96% Sens CIN3  90%  88%  88%  90%  90% Sens CIN2  67%  64%  64% 67%  67% Spec Cntrl  93%  91%  88%  84%  88% Spec CIN0  86%  84%  88% 86%  81% Spec CIN1  73%  66%  73%  68%  66% CIN0 test− 37 36 38 37 35CIN0 test+  6  7  5  6  8 CIN1 test− 30 27 30 28 27 CIN1 test+ 11 14 1113 14 CIN2 test− 14 15 15 14 14 CIN2 test+ 28 27 27 28 28 CIN3 test−  4 5  5  4  4 CIN3 test+ 38 37 37 38 38 cncr test−  2  3  0  0  3 cncrtest+ 80 79 82 82 79 ctrl test− 64 63 61 58 61 ctrl test+  5  6  8 11  8Overall  88%  86%  88%  89%  87% sens Overall  86%  82%  84%  80%  80%spec

As cytology is currently been used and hr-HPV testing has been suggestedas primary screening tool in population-based cervical screening, wesimulated the effect on the performances of the methylation tests ifonly cytology (Table 13) or hr-HPV (Table 14) positive patients wereselected. The triage simulations were based on the performance resultsobtained in Table 11 and Table 12. The performance of cytology andhr-HPV testing were based on data from literature.

The performances of the triage tests showed much higher specificityresulting in fewer referrals for colposcopy than did cytology or hr-HPVtesting alone but were less sensitive. Testing for hr-HPV types has ahigher sensitivity for detecting CIN2+ than cytology. The NPV is closeto 100% thus allowing for less frequent screening and longer screeningintervals without jeopardizing patients' safety. But, the enthusiasm forusing HPV testing in primary screening has been tempered by its somewhatpoorer PPV (19%) in comparison with cytological analysis (27%). Usingmethylation as triage test, the PPVs were much higher.

Taking the limitations of cytology and the decreased disease prevalencedue to the introduction of HPV vaccination programs into account, it isproposed to use a highly sensitive and objective screening test such asHPV DNA testing to identify the rare cases of cancer precursors and tocombine it, when positive, with another test which has a high degree ofspecificity, such as methylation testing. Moreover, methylation ismeasuring changes in the host cells, as precursor of cervix cancer,while HPV is detecting the causative agent. This is an ideal methodologyfor a screening and a triage assay because they should measure differentbut complementary biological signals.

TABLE 13 The simulation of the performance of Cytology test as afirst-line screening test on 70000 women and the methylation markertest(s) in-or excluding HPV16 as triage test. (Sens: sensitivity; Spec:specificity; CIN0/1, CIN2+: cervical intraepithelial neoplasia grade 0and 1, and grade 2 and 3 and cancers; PPV: positive predictive value;NPV: negative predictive value). Cytology, Cytology, Cytology, Cytology,Triage Cytology, Cytology, Triage Triage Triage NID2\ Triage hr- TriageJAM3\ JAM3\ NID2\ LMX1A\ Cytology HPV HPV16 NID2 NID2\HPV16 LMX1A HPV16CIN2+ Test+ 540 449 334 213 417 290 417 CIN2+ Test− 230 321 436 557 353480 353 CIN0/1 Test+ 1460 825 219 85 288 187 374 CIN0/1 Test− 6777068405 69011 69145 68942 69043 68856 Sens 70.1% 58.3% 43.4% 27.6% 54.2%37.7% 54.2% Spec 97.9% 98.8% 99.7% 99.9% 99.6% 99.7% 99.5% NPV 99.7%99.9% 99.7% 99.5% 99.8% 99.6% 99.8% PPV 27.0% 35.2% 60.4% 71.4% 59.1%60.8% 52.7% Colposcopy 2000 1274 553 298 706 477 791 referrals

TABLE 14 The simulation of the performance of hr-HPV test as afirst-line screening test on 70000 women and the methylation markertest(s) in-or excluding HPV16 as triage test. (Sens: sensitivity; Spec:specificity; CIN0/1, CIN2+: cervical intraepithelial neoplasia grade 0and 1, and grade 2 and 3 and cancers; PPV: positive predictive value;NPV: negative predictive value). hr-HPV, hr-HPV, hr-HPV, Triage hr-HPV,Triage hr-HPV, hr-HPV, Triage JAM3\ Triage NID2\ Triage Triage JAM3\NID2\ NID2\ LMX1A\ hr-HPV Cytology HPV16 NID2 HPV16 LMX1A HPV16 CIN2+Test+ 665 532 433 276 541 376 541 CIN2+ Test− 67 285 333 491 226 390 226CIN0/1 Test+ 2835 553 420 164 553 358 717 CIN0/1 Test− 66433 68631 6881469070 68681 68875 68517 Sens 90.8% 65.1% 56.5% 36.0% 70.6% 49.1% 70.6%Spec 95.9% 99.2% 99.4% 99.8% 99.2% 99.5% 99.0% NPV 99.9% 99.6% 99.6%99.4% 99.8% 99.5% 99.8% PPV 19.0% 49.0% 50.8% 62.7% 49.5% 51.2% 43.0%Colposcopy 3500 1085 853 440 1094 735 1258 referrals

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The disclosure of each reference cited in this disclosure is expresslyincorporated herein.

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The invention claimed is:
 1. A method for identifying, in a test sample,cervical cancer or its precursor, or predisposition to cervical cancerand performing screening or treating, said method comprising: providinga test sample comprising cervical cells or nucleic acids from cervicalcells; detecting in said test sample methylation of at least one geneselected from the group consisting of EPB41L3, JAM3, and TERT bycontacting at least a portion of the gene or promoter region thereof ofthe test sample with a chemical reagent that selectively modifies anon-methylated cytosine residue relative to a methylated cytosineresidue, or selectively modifies a methylated cytosine residue relativeto a non-methylated cytosine residue; and detecting a product generateddue to said contacting, the product obtained by contacting at least aportion of the TERT gene or promoter region thereof comprising SEQ IDNO:384; wherein methylation in said test sample indicates the presenceof cells that are neoplastic, precursor to neoplastic, or predisposed toneoplasia, or the presence of nucleic acids from cells that areneoplastic, precursor to neoplastic, or predisposed to neoplasia,wherein if the at least one gene is methylated, refer the woman forcolposcopy; or if the at least one gene is unmethylated, refer the womanto screening for the presence of hr-HPV; or if the at least one gene ismethylated, refer the woman for cervical resection.
 2. The method ofclaim 1, further comprising detecting in said test sample themethylation of C13orf18.
 3. The method according to claim 1, wherein thetest sample comprises squamous cells, nucleic acids from squamous cells,adenocarcinoma cells, nucleic acids from adenocarcinoma cells,adenosquamous cell carcinoma cells, nucleic acids from adenosquamouscarcinoma cells, or any combination thereof.
 4. The method according toclaim 1, wherein the test sample is from a specimen selected from thegroup consisting of a tissue specimen, a biopsy specimen, a surgicalspecimen, a cytological specimen, cervical scrapings, cervical smear,cervical washing, vaginal excretions, and blood.
 5. The method of claim4, wherein the test sample is from a biopsy specimen and surgicalremoval of neoplastic tissue is recommended to the patient.
 6. Themethod of claim 4, wherein the specimen is a surgical specimen andadjuvant chemotherapy or adjuvant radiation therapy is recommended tothe patient.
 7. The method according to claim 1, wherein epigeneticmodification of at least two genes is detected.
 8. The method accordingto claim 1, wherein said methylation is at a CpG dinucleotide motif inthe gene, or a promoter region thereof.
 9. The method of claim 1,wherein the step of detecting a product employs amplification with atleast one primer that hybridizes to a sequence comprising a modifiednon-methylated CpG dinucleotide motif but not to a sequence comprisingan unmodified methylated CpG dinucleotide motif thereby formingamplification products.
 10. The method of claim 1, wherein the step ofdetecting a product comprises amplification with at least one primerthat hybridizes to a sequence comprising an unmodified methylated CpGdinucleotide motif but not to a sequence comprising a modifiednon-methylated CpG dinucleotide motif thereby forming amplificationproducts.
 11. The method of claim 1, wherein the product is detected bya method selected from the group consisting of electrophoresis,hybridization, amplification, sequencing, ligase chain reaction,chromatography, mass spectrometry, and combinations thereof.
 12. Themethod of claim 1, wherein the chemical reagent comprises bisulfiteions.
 13. The method of claim 12, further comprising treating thebisulfite ion-contacted, at least a portion of the gene with alkali. 14.The method according to claim 1, wherein the step of detecting employsamplification of at least a portion of the at least one gene using anoligonucleotide primer that specifically hybridizes under amplificationconditions to a region of a gene selected from the group consisting ofEPB41L3, JAM3, and TERT; wherein the region is within about 10 kb ofsaid gene's transcription start site.
 15. The method according to claim1, wherein the step of detecting employs amplification of at least aportion of the at least one gene using at least one pair ofoligonucleotide primers that specifically hybridizes under amplificationconditions to a region of a gene selected from the group consisting ofgenes according to EPB41L3, JAM3, and TERT; wherein the region is withinabout 10 kb of said gene's transcription start site.
 16. The method ofclaim 15, further comprising one pair of oligonucleotide primers thatspecifically hybridize under amplification conditions to C13orf18.
 17. Amethod for identifying, in a test sample, cervical cancer or itsprecursor, or predisposition to cervical cancer and performing screeningor treating, said method comprising: providing a test sample comprisingcervical cells or nucleic acids from cervical cells; detecting in saidtest sample methylation of at least one gene selected from the groupconsisting of EPB41L3, JAM3, and TERT by contacting at least a portionof the gene or promoter region thereof of the test sample with achemical reagent that selectively modifies a non-methylated cytosineresidue relative to a methylated cytosine residue, or selectivelymodifies a methylated cytosine residue relative to a non-methylatedcytosine residue; and detecting a product generated due to saidcontacting, the product comprising at least one of SEQ ID NO:309, SEQ IDNO:310, SEQ ID NO:331, and SEQ ID NO:384; wherein methylation in saidtest sample indicates the presence of cells that are neoplastic,precursor to neoplastic, or predisposed to neoplasia, or the presence ofnucleic acids from cells that are neoplastic, precursor to neoplastic,or predisposed to neoplasia, wherein if the at least one gene ismethylated, refer the woman for colposcopy; or if the at least one geneis unmethylated, refer the woman to screening for the presence ofhr-HPV; or if the at least one gene is methylated, refer the woman forcervical resection.
 18. The method of claim 17, further comprisingdetecting in said test sample the methylation of C13orf18.
 19. Themethod of claim 17, wherein the test sample is from a biopsy specimenand surgical removal of neoplastic tissue is recommended to the patient.20. The method of claim 17, wherein the specimen is a surgical specimenand adjuvant chemotherapy or adjuvant radiation therapy is recommendedto the patient.
 21. A method for identifying, in a test sample, cervicalcancer or its precursor, or predisposition to cervical cancer andperforming screening or treating, said method comprising: providing atest sample comprising cervical cells or nucleic acids from cervicalcells; detecting in said test sample methylation of at least one geneselected from the group consisting of EPB41L3, JAM3, and TERT bycontacting at least a portion of the gene or promoter region thereof ofthe test sample with a chemical reagent that selectively modifies anon-methylated cytosine residue relative to a methylated cytosineresidue, or selectively modifies a methylated cytosine residue relativeto a non-methylated cytosine residue; and detecting a product generateddue to said contacting by amplifying the contacted portion and detectingan amplification product, the amplification product comprising at leastone of SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:331, and SEQ ID NO:384;wherein methylation in said test sample indicates the presence of cellsthat are neoplastic, precursor to neoplastic, or predisposed toneoplasia, or the presence of nucleic acids from cells that areneoplastic, precursor to neoplastic, or predisposed to neoplasia,wherein if the at least one gene is methylated, refer the woman forcolposcopy; or if the at least one gene is unmethylated, refer the womanto screening for the presence of hr-HPV; or if the at least one gene ismethylated, refer the woman for cervical resection.
 22. The method ofclaim 21, wherein the step of detecting a product comprises amplifyingthe contacted portion with at least one primer that hybridizes to asequence comprising a modified non-methylated CpG dinucleotide motif butnot to a sequence comprising an unmodified methylated CpG dinucleotidemotif thereby forming amplification products.
 23. The method of claim21, wherein the step of detecting a product comprises amplifying thecontacted portion with at least one primer that hybridizes to a sequencecomprising an unmodified methylated CpG dinucleotide motif but not to asequence comprising a modified non-methylated CpG dinucleotide motifthereby forming amplification products.